Current view on the structure-function relationship of postsynaptic neurotoxins from snake venoms

Pharmac. Ther,Vol. 34, pp. 403 to 451, 1987 Printed in Great Britain. All rights reserved

0163-7258/87 $0.00+0.50 Copyright © 1987 Pergamon Journals Ltd

Specialist Subject Editor: A. L. H ~ w Y

CURRENT VIEW ON THE STRUCTURE-FUNCTION RELATIONSHIP OF POSTSYNAPTIC NEUROTOXINS FROM SNAKE VENOMS TOSHIYA ENDO* and NUBUO TAMIYA~" *Department of Chemistry, College of Technology, Ganma University, Kiryu 376, Japan "fDepartment of Chemistry, Faculty of Science, Tohoku University, Aobayama, Sendal 980, Japan

1. INTRODUCTION Out of about 2,340 species of snakes living on the earth, more than 420 species are venomous (Dowling and Duellman, 1978). The venoms of proteroglyphous or fixed-frontfanged snakes (about 240 species) arc extremely toxic. The proteroglyphous snake venom.s contain various toxic components, and among the most toxic are proteins of postsynaptic neurotoxins or a-neurotoxins, which are usually referred to simply as neurotoxins. The neurotoxin binds to the nicotinic acetylcholine receptor (AChR) in the postsynapfic membranes of skeletal muscles, prevents the binding of chemical neurotransmitter acetylcholine and thereby blocks the excitation of muscles. This block at the neuromuscular junction leads to flaccid paralysis (reviewed in Chang, 1979). As this pharmacological action is similar to that of curare (tubocurarine), an alkaloid arrow poison used by South American Indians (except for the higher affinity of neurotoxins for the receptor), neurotoxins are also called curaremimetic neurotoxins. Although snake venoms have for a long time been the subject of scientific investigation with much fascination because of their strong and distinct action, it was at the end of the 1950s to early 1960s that the neurotoxins were isolated in a rather pure form from cobras ($asaki, 1957) and sea snakes (Carey and Wright, 1960). In the mid-60s, neurotoxins in pure form were isolated and some properties were characterized (Yang, 1965; Tamiya and Arai, 1966; Karlsson et al., 1966). A few years later, the amino acid sequence of a neurotoxin was determined for the first time: for toxin a (Tx a) from the spitting cobra, Naja nigricollis, by Eaker and Porath (1967). Reports on the amino acid sequences for a number of homologous neurotoxins from such snakes as cobras, kraits, mambas, Australian elapid, sea kraits and sea snakes then followed. In the 1970s, extensive chemical modification studies were carried out with neurotoxins (reviewed in Karlsson, 1979) in parallel with the comparison of growing sequence data, aiming at the elucidation of the relation between specific amino acid residues in the sequences and the function of neurotoxins. Several residues that are important for expressing neurotoxicity were identified at this stage. The next important step for understanding the function of neurotoxins in terms of molecular structures was brought about by the determination of the crystal structure of erabutoxin b (Eb), a neurotoxin from a sea krait, Laticauda semifasciata (Low et al., 1976; Tsernogiou and Petsko, 1976). This allowed the re-interpretation of the previous sequence data and chemical modification results for neurotoxins on the basis of the three-dimensional structure and the visualization of a possible reactive site of the neurotoxin for the receptor. Then from the late 1970s to 1980s, efforts were made to elucidate various conformational aspects of neurotoxins in solution and to confirm that the deduced conformation of Eb occurs in other neurotoxins in solution (reviewed in Dufton and Hider, 1983). These studies include nuclear magnetic resonance (NMR) analysis of neurotoxins in solution and X-ray diffraction analyses on the crystal structures of two other neurotoxins, a-cobratoxin (a-Cbt) from Thai cobra, 4o3

404

T. ENDOand N. TAMIYA

Naja naja siamensis (Walkinshaw et al., 1980), and ~-bungarotoxin (~t-Bgt) from banded krait, Bungarus multicinctus (Agard and Stroud, 1982). The accumulated knowledge on the structure of neurotoxins aided the understanding of the immunology of neurotoxins at the level of molecular structure, which is important because of the serious demand for therapeutics for venomous snake bites (see Mrnez, 1985). The highly selective action of neurotoxins on the nicotinic acetylcholine receptor also contributed to the rapid progress in the pharmacological and biochemical characterization of the receptor since the early 1970s. Indeed, the nicotinic acetylcholine receptor is at present the best characterized neurotransmitter receptor (reviewed by Conti-Tronconi and Raftery, 1982; Changeux et al., 1984; McCarthy et al., 1986). The aim of the present article is to give details of the recent developments in the research on the structure-function relationship of neurotoxins, especially after the appearance of the crystal structure of the neurotoxins, and also to provide complements to understanding the function of the acetylcholine receptor from the viewpoint of neurotoxin-receptor interaction and its functional consequences. 2. SEQUENCE 2.1. PRIMARYSTRUCTURE In 1967, Eaker and Porath reported the first amino acid sequence of a neurotoxin, Tx ~ (Naja nigricollis). Since then, a large number of neurotoxins have been isolated from proteroglyphous snakes, families Elapidae and Hydrophiidae (the classification here follows that of Smith et al., 1977, and Mengden, 1983, as shown in Table 1), and sequenced. Previous sequence analyses of neurotoxins have covered four subfamilies of proteroglyphous snakes (subfamilies Bungarinae and Elapinae of family Elapidae and subfamilies Oxyuraninae and Hydrophinae of family Hydrophiidae). At present, amino acid sequences of about 80 neurotoxins are available, and they form one of the largest families of proteins with known primary structures. The sequence data of neurotoxins are compiled in Table 2, where alignment is made with appropriate gaps to achieve maximum sequence homology. Weak neurotoxins that show reduced neurotoxicity, and neurotoxin homologs (whose sequences are related to those of neurotoxins) with generally unknown pharmacological activity are also listed in the table. Some of the neurotoxin homologs from the genus Dendroaspis (mamba) are often referred to as angusticeps-type toxins (Viljoen and Botes, 1973, 1974). Recently, two of these have been shown to inhibit acetylcholinesterase (Karlsson et al., 1986). Throughout this review, the numbering system and abbreviations for the names of neurotoxins are used as shown in Table 2. According to the sequences, neurotoxins may be classified into two subgroups termed short neurotoxins and long neurotoxins. Short neurotoxins contain 60 to 62 amino acid residues, and 4 disulfide bridges in common positions, beween Cys-3 and Cys-24, Cys-17 and Cys-45, Cys-49 and Cys-60, and Cys-61 and Cys-66. Most of the long neurotoxins consist of 70 to 74 amino acid residues and have the fifth disulfide bridge between Cys-30 and Cys-34 in addition to the four disulfide bridges common to short neurotoxins. In comparison with short neurotoxins, long neurotoxins have further characteristic features with respect to the sequences: the deletion of amino acid residues in the segment of residues 4 to 16; the presence of extra amino acid residues 32, 34, 35 and 36; the insertion of the sequence Ala-46, Ala-47 and Thr-48 between Cys-45 and Cys-49 instead of Gly-48 in short neurotoxins; and the presence of an extra "C-terminal tail" segment with 5 to 9 amino acid residues. An exceptional long neurotoxin with respect to the size of the molecule is Ls III (Laticauda semifasciata), which consists of 66 amino acid residues and has an extra C-terminal tail with only two residues. Two other exceptional long neurotoxins are Lc a and L c b from Laticauda colubrina, which consist of 69 amino acid residues, and have Aspo30 and Gly-34 instead of half cystine residues. Thus, they lack the fifth disulfide bridge commonly found in other long neurotoxins. However, Ls III, Lc a and Lc b have the other characteristic features of long neurotoxins (Table 2). Thus we accept Ls III, Lc a

f

TABLE 1. Classification of Proteroglyphous (Elapid) Snakes (Mengden, 1983). When Common Names are Available, They Are Shown in Parentheses. Subfamily Tribe Genus Bungarus (Kraits) Bungarini (Asiatic cobras) Aspidelaps (Shield-nosed cobras), Boulengerina (Water cobras), Bungarinae Dendroaspis (Mambas), Elapsoidea (African garter snakes), Najini (Cobras) Hemachatus (Ringhals Cobras), Naja (Cobras), Ophiophagus (King (Afroasian cobras) Cobra), Paranaja (Burrowing Cobra), Pseudohaje (Tree cobras), Waherinnesia (Desert Cobra) Elapidae I Elapini Calliophis (Asian coral snakes), Leptomicrurus (Coral snakes), Micruroides (Arizona Coral Snake), Micrurus (Coral snakes) (American and North Asiatic coral snakes) Maticora (Coral snakes), Parapistocalamus (Hediger's Snake) Maticorini Elapinae (South Asiatic coral (Coral snakes) snakes) Laticauda (Sea kraits) Laticaudini (Sea kraits) "Acanthophis (Death adders), Aspidomorphus, Austrelaps (Copperhead), Cacophis (Crowned snakes), Cryptophis (Small-eyed snakes), Demansia (Whip snakes), Denisonia, Drysdalia (Whitelipped snakes), Echiopsis (Desert Snake), Elapognathus (Little Brown Snake), Furina (Red-naped Snake), Glyphodon, Hemiaspis •Oxyuraninae (Marsh Snake), Hoplocephalus (Broad-headed snakes), Loveridgelaps (Terrestrial palatine (Banded Small-eyed Snake), Micropechis (New Guinea Small-eyed draggers) Snake), Neelaps, Notechis (Tiger snakes), Ogmodon, Oxyuranus (Taipans), Pseudechis (Black snakes and King Brown), Pseudonaja (Brown snakes), Rhinoplocephalus (Muller's Snake), Salomonelaps, Hydrophiidae Simoselaps (Desert banded snakes), Suta (Curl snakes), (Palatine draggers) Toxicocalamus, Tropidechis (Rough-scaled snake), Unechis (Blackheaded snakes), Vermicella (Bandy-bandy) (" Ephalophiini Ephalophis, Parahydrophis Hydrelaps Hydrophiinae ~ Hydrelapini Aipysurus, Emydocephalus (True sea snakes) | Aipysurini ~ Acalyptophis, Astrotia, Disteira, Enhydrina, Hydrophis, Kerilia, [. Hydrophiini Kolpophis, Lapemis, Thala~sophis, Pelamis (Pelagic sea snake)

Family

406

T. ENDOand N. TAMIYA

and L c b as long neurotoxins, and accordingly the following definition should be adopted for long neurotoxins: long neurotoxins consist of 66 to 74 amino acid residues with 4 or 5 disulfide bridges. Ls III has weak toxicity (Maeda and Tamiya, 1974), but Lc a and Lc b have normal toxicity (Kim and Tamiya, 1982). The large data set of the amino acid sequences of neurotoxins is naturally a matter of interest from the viewpoint of taxonomy and evolution of snakes. In particular, the sequences can be expected to provide a reliable basis for the presently rather confused taxonomy of the proteroglyphous snakes. One species of snake often has more than one neurotoxin and often has both short and long neurotoxins. Sequence homology analyses •may be carried out for short and long neurotoxins independently, and then the results may be compared with each other or with those obtained from the morphological analyses. Difference matrix analyses of the amino acid sequences (Fitch and Margoliash, 1967) has been applied to neurotoxins for the classification of proteroglyphous snakes (Tamiya and Yagi, 1985). The sequences of short neurotoxirts suggest that they are classified into four groups: those of sea kraits (Laticauda); those of true sea snakes (Astrotia, Pelamis, Enhydrina, Hydrophis, Lapernis, and Aipysurus) and Australian elapids (Pseudechis and Acanthophis); those of cobras (Naja and Haemachatus); and those of mambas (Dendroaspis). No short neurotoxins are found from kraits (Bungarus). Long neurotoxins can be classified into six groups: those of sea kraits (Laticauda), those of true sea snakes (Astrotia); those of Australian elapids (Acanthophis and Notechis), those of cobras (Ophiophagus and Naja); those of kraits (Bungarus); and those of mambas (Dendroaspis). Short neurotoxins from Australian elapids (Oxyuraninae: Pseudechis and Acanthophis), Pa a (Pseudechis australis) and Aa c (Acanthophis antarcticus), have cysteine residues in positions 3 and 4, and Cys-3 forms a disulfide bond with Cys-24 (Table 2). Appearance of cysteine residues in positions 3 and 4 are also observed for short neurotoxins from true sea snakes (Hydrophiinae), and this is consistent with the classification shown in Table 1, in which Oxyuraninae and Hydrophiinae are subfamilies of the same family, Hydrophiidae. In the case of long neurotoxins, the homology within each group is lower than in the case of short neurotoxins, probably because a smaller number of sequences are available for long neurotoxins at present. The classifications deduced from the sequence analyses of short and long neurotoxins are in accord with each other and with that from morphological analyses in the most part, but at the same time there are some discrepancies among them. Failure to superimpose these classifications precisely on one another has led to the proposal of "a non-divergence theory of evolution" (Tamiya and Yagi, 1985). This theory is based on the observation that comparison of the amino acid sequences of related proteins (short and long neurotoxins) in various organisms (snakes) gives inconsistent results from one type of protein to another. Concerning the driving force of evolution, it focuses more attention on information exchange among the species through hybridization and infection rather than on mutation within the isolated species. Strydom (1979) has constructed a phylogenetic tree of neurotoxins and claimed that long neurotoxins evolved from the ancestor of cardiotoxins without neurotoxicity and then short neurotoxins appeared. In contrast, Dufton (1984) has, on the basis of a phylogenetic analysis of neurotoxins and cardiotoxins, pointed out that short neurotoxins resemble cardiotoxins more closely than do long neurotoxins. However, it should be noted that the non-divergence theory of evolution mentioned above poses a question about such phylogenetic analyses (Tamiya and Yagi, 1985). Recently, cDNA of Ea (Laticauda semifasciata) has been cloned and sequenced (Tamiya et al., 1985). It is led by a sequence encoding a hydrophobic peptide fragment, Met-Lys-Thr-Leu-Leu-Leu-Thr-Leu-Val-Val-Val-Thr-Ile-Val-Cys-Leu-Asp-Leu-GlyTyr-Thr, which probably constitutes a signal sequence involved in the secretion process of the neurotoxin. One noteworthy point is that a valine-repeating sequence, Val-Val-ValThr-Ile-Val, is found in the hydrophobic part of the signal sequence. Such a valine-rich sequence does not favour a helical conformation (Katakai et al., 1973) that is assumed to be an active conformation of the signal sequence on passage through the lipid membrane.

T~L~ 2. AmO:oAcid Sequences of Neurotoxina and Neaotoxin Homolo8s. (1-45) Short Neurotoxins; (46~0) Long Lourowxins; (81)

Receptor: (82, lOJ and 104) Weak Neurotoxim and (83-102,105 and 106)Neurotoxi~ Homologs with Unknown PharmacologicalAc#oify (17) and (18) are Identical with (16), (20) Identical with (19), and (34) and (35) Nm'etoxla"

A,t~ao ~kl r a k ~

.... + .... 1.... + .... 2 .... + .... 3 .... + .... 4 .... + .... 3-+ 6 + 7 GCPTVKPGIKLRCCESEDCNN RGSITERGC ]~ll R R C Y N Q Q S S Q P K T T K S C P P G E N S C Y N K ( WRD H GCPKVKPGIKLRCCESEDCNN RGSITERGC 2~c R R C Y N Q Q S S Q P K T T K S C P P G E N S C Y N K ( WRD H GCPKVKPG]KLRCCESEDCNN RGSITERGC 3~d R R C F N Q Q S S Q P K T T K S C P P G E N S C Y N K ( WRD H GCPTVKPGIKLTCCQSEDCNN RGTIIERGC 4Ua(Am)t R R C F N H P S S Q P Q T N K S C F P G E N S C Y N K ( WRD H G CPTVKPGIKLTCCQSEDCNN H R G T I T E R G C 5UB~" R R C F N H P S S Q P Q T N K S C P P G E N S C Y N K ( WRD GCPQVKSGIKLTCCQSDDCNN RGTITERGC hUb(Amp R R C F N H P S S Q P Q T N K S C P P G E N S C Y N K ( WRD H G C PTVKPGVKLRCCQSEDCNN H R G T I I E R G C 7Llc(Amp R R C F N Q Q S S Q P Q T N K S C P P G E N S C Y R K ( W R D GCPTVKPGIKLTCCQSDDCNN RGTITERGC RRCFNHPSSQPQTNKSCPPGENSCYNK(WRD H 8Lcrao G C PQVKSG1KLTCCQSDDCNN H RGTI ERGC R R C F N H F S S Q F Q T N K S C P P G E N S C Y N K ( W R D 9Lcrb~ GCPTVKPGIKLRCCQSEDCNN RGTI ERGC RRCFNQQSSQPQTNKSCPPGENSCYRK(WRD H 10 Lcrc" GCPTVKPGIKLSCCESEVCNN RGTI ERGC RICFNHQSSQFQTTKTCSPGESSCYNKtWSD F llEa GCPTVKPG]KLSCCESEVCNN RGTI ERGC RICFNHQSSQPQTTKTCSPGESSCYHK(WSD F 12 Eb GCPTVKPG]NLSCCESEVCNN R G T I ERGC RICFNHQSSQFQTTKTCSPGESSCYHK(WSD F 13 E¢ GCPQVKSG1KLECCHTNECNN RGTI ERGC MTCCNQQSSQPKTTTNCA GNSC.YKKTWSD H 14Asa GCPQVKKGIKLECCHTNECNN RGTR ERGC MTCCNQQSSQPKTTTNCA ESSCYKKTWSD H 15 Hca GCPQVKSGIKLECCHTNECNN RGTR1ERGC MTCCNQQSSQPKTTTNCA ESSCYKKTWSD H 16 He b

+

8

17 Es 5 18 Fix a

19 Es4 20 Lh 21 HI a 22Ala 23 Alb 24Aic 23 Paa 26~c 27~kVn.ll ~4.11.3 ~Dpp~ 30 Nm d 3[ ~ 32 Nnv 33 Nnv G M N~6 35 N ~ ~N~I~ 37HhlI 3GHhlV ~t Nnp 41Nnoll 42Nmml 43Nmmlll N~I0 45Nha14 ~a 47~b ~Ill ~ ~ As~ ~l~b 52N~I114 53 ~ I M ~V 55 ~ h,9.3 56 ~4.7,3c 571~V~ 5gDpp 7

MTCCNQQSSQPKTTTNCA

ESSCYKKTWSD H

MTCCNQQSSQPKTTTNCA ESSCYKKTWRD LTCCNQQSSQPKTTTDCA DNSCYKKTWQD LTCCNQQSSQPKTTTDCA DNSCYKMTWRD LTCCNQQSSQPKTTTDCA DNSCYKKTWKD MTCCNQQSSQPKTTTICAGGESSCYKKTWSD MQCCNQQSSQPKTTTTCPGGVSSCYKKTWRD RICYNHQSTTPATTKSC GENSCYKKTWSD RICYNHQSTTPATTKSC GENSCYKKTWSD RICYNHQSTTRATTKSC EENSCYKKYWRD M E C H N Q Q S S Q P P T T K T C P GETNCYKKQWSD L E C H N Q Q S S Q P P T T K T C P GETNCYKKVWRD M I C H N Q Q S S Q R P T I K T C P GETNCYKKRWRD L E C H N Q Q S S Q P P T T K T C P GETNCYKKRWRD

F H H H H H H H H H H H H

RGTRIERGC

GCPQVKPGIKLECCHTNECNN

RGTRIERGC RGTRIERGC RGTRIERGC RGTRIERGC RGSRTERGC RGTIIERGC RGTIIERGC RGTIIERGC RGTIIERGC RGTIIERGC RGTIIERGC RGTIIERGC RGSITERGC

GCPQVKPG[KLECCHTNECNN GCPQVKPG[KLECCKTNECNN GCPQVKPGIKLECCKTNECNN GCPQVKPG[KLECCKTNECNN GCPHVKPGIKLTCCKTDECNN GCPRVKPGIRLICCKTDECNN GCPKVKQGIHLHCCQSDKCNN GCPKVKRGVHLHCCQSDKCNN GCPKVKPGVGIHCCQSDKCNY GCPSVKKGVKINCCTTDRCNN GCPTVKPGIKLNCCTTDKCNN GCPSVKKGVGIYCCKTDKCNR GCPSVKKGIEINCCTTDKCNN

RGTIIERGC GCPSVKKGVGIYCCKTDKCNR M I C H N Q Q S S Q P P T I K T C P GETNCYKKQWRD H RGTIIERGC GCPTVKPGINLKCCTTDRCNN L E C H N Q Q S S Q F F T T K S C P GDTNCYNKRWRD H RGSRTERG~ GCPTVKPGIKLKCCTTDRCNK L E C H N Q Q S S Q T P T T Q T C P GETNCYKKQWSD H H RGYRTERGC GCPSVKNGIEINCCTTDRCNN LECHNQQSSQTPTTTGCSGGETNCYKKRWRD RGTIIERGC GCPKVKPGVKLNCCRTDRCNN L E C H N Q Q S S Q A P T T K T C S GETNCYKKWWSD H RGTIIERGC GCPKVKPGVNLNCCRTDRCNN L E C H N Q Q S S Q P P T T K T C S GETNCYKKWWSD H RGYRTERGC GCPTVKKGIELNCCTTDRCNN LECHNQQSSEPPTTTRCSGGETNCYKKRWRD H RGYKTERGC GCPTVKKGIQLHCCTSDNCNN LNCHNQMSAQPPTTTRCSRWETNCYKKRWRD H RGTllERGC GCPSVKKG]EINCCTTDKCNR M I C Y K Q Q S L Q F P l T T V C P GEKNCYKKQWSG H RGTIIERGC GCPSVKKGVGIYCCKTNKCNR M I C H N Q Q S S Q P P T I K T C P GETNCYKKRWRD H LAP RDT Q I C A P G Q E I C Y L K S W D D G T G F L K G N R L E F G C A A T C P T V K P G I D I K C C S T D K C N P H P K L A RICY LAP RDT Q I C A P G Q E I C Y L K S W D D G T G S 1 R G N R L E F G C A A T C P T V K R G I H I K C C S T D K C N P H P K L A RICY LNP HDT Q T C P S G Q E I C Y V K S W C N A W C S S R G K V L E F G C A A T C P S V N T G T E I K C C S A D K C N T Y P RECY LGY K H S Q T C P P G E N V C F V K T W C D G F C N T R G E R I I M G C A A T C P T A K S G V H I A C C S T D N C N I Y A K W G S LSCY LGY K H S Q T C P P G E N V C F V K T W C D A F C S T R G E R [ V M G C A A T C P T A K S G V H I A C C S T D N C N I Y T K W G S G R LSCY RGY NN P Q T C P P G E N V C F T R T W C D A F C S S R G K V V E L G C A A T C P ] V K S Y N E V K C C S T D K C N P F P V R P R R P P VICY MGP K T P R T C P R G Q N L C Y T K T W C D A F C S S R G K V V E L G C A A T C P I A K S Y E D V T C C S T D N C N P F P V R P R H P P LICY KTP S V K P E T C P H G E N I C Y T E T W C D A W C S Q R G K R V E L G C A A T C P K V K A G V G I K C C S T D N C N P F P V W N P R G RTCY KTP S V K F E T C P H G E N I C Y T E T W C D A W C S Q R G K R V E L G C A A T C P K V K A G V G I K C C S T D N C N P F P V W N P R RICY KTP S V K P E T C P H G E N I C Y T E T W C D A W C S Q R G K R E E L G C A A T C P K V K A G V G I K C C S T D N C D P F P V K N P R RTCY KTP S V K P E T C P H G E N I C Y T E T W C D A W C S Q R G K R E E L G C A A T C P K V K A G V G I K C C S T D N C D P F F V K N P R RTCY KTF S D Q S K I C P P G E N I C Y T K T W C D A W C S Q R G K I V E L G C A A T C P K V K A G V E I K C C S T D N C N L F K F G K P R RTCN KTF S D Q S K I C P P G E N I C Y T K T W C D A W C S Q R G K R V E L G C A A T C P K V K A G V E I K C C S T D D C D K F Q F G K P R RTCN KTP SDQSK1CPPGENICYTKTWCDAWCSQRGK]VELGCAATCPKVKAGVE]KCCSTDNCNKFKFGKPR 59D~pG RTCN KTY S D K S K T C P R G E N I C Y T K T W C D G F C S Q R G K R V E L G C A A T C P K V K T G V E I K C C S T D Y C N P F P V W N P R ~kVn-llll RTCY VTP D V K S Q T C P A G Q D I C Y T E T W C D A W C T S R G K R V N L G C A A T C P I V K P G V E I K C C S T D N C N P F F T W R K R P TKCY 61 Oha VTP D A T S Q T C P D G Q D I C Y T K T W C D G F C S S R G K R I D L G C A A T C P K V K P G V D I K C C S T D N C N P F P T W K R K H 62 Ohb TKCY VTP D V K S E T C P A G Q D L C Y T D T W C V A W C T V R G K R V S L G C A A I C P I V P P K V S I K C C S T D A C G P F P T W P N V R 63 Oh9 TKCY ITP DVTSQACPDGQNICYTKTWCDNFCGMRGKRVDLGCAATCPTVKPGVDIKCCSTDNCNPFPTRERS M Nha Ell IRCF ITP DVTSQACPDGH VCYTKMWCDNFCGMRGKRVDLGCAATCPTVKPGVDIKCCSTDNCNPFPTRKRS 65 Nhh 5 IRCF iTF DVTSQICADGH VCYTKTWCDNFCASRGKRVDLGCAATCPTVKPGVNIKCCSTDNCNPFPTRNRP 66 Nm b IRCF RTP DLKSQTCPPGEDLCYTKKWCDAWCTSRGKV1ELGCVATCPKVKPYEQITCCSTDNCNPHPKMKP 67 Nm 3.9,4 KRCY ITP DVTSQACPAGH VCYTKMWCDNFCGMRGKRVDLGCAATCPTVKPGVDIKCCSTDNCNPFPTRKRS 68 Nhh D*VI1 I R C F ITF DVTSQACFDGH VCYTKMWCDNFCGMRGKRVDLGCAATCPKVKPGVN1KCCSRDNCNPFPTRKRS 69 Nnv~t IRCF I T P D I T S K D C P N G H VCYTKTWCDAFCS R G K R V D L G C A A T C P T V K T G V D I Q C C S T D N C N P F F T R K R P 70 ~-C'bt IRCF ITP DITSKDCPNGH VCYTKTWCDGFCS]RGRVDLGCAATCPTVRTGVDIQCCSTDNCNPFPTRKRP 71 Nnn 3 RCF [TP DITSKDCPNGH VCYTKTWCDGFCSSRGKRVDLGCAATCPTVRTGVDIQCCSTDNCNPFPTRKRP 72 Nnn 4 RCF ITP DITSKDCPNGH VCYTKTWCDGFCSIRGKRVDLGCAATCPTVKTGVDIQCCSTDNCNPFPTRKRP 73 Nnn 3WP RCF ITP DITSKDCPNGH VCYTKTWCDGFCSIRGKRVDLGCAATCPTVRTGVD]QCCSTDDCDPFPTRKRP 74TxA RCF ITP DITSKDCPNGH VCYTKTWCDGFCSSRGKRVDLGCAATCPTVRTGVDIQCCSTDDCDPFPTRKRP 75TxB RCF ITP DITSKDCPNGH VCYTKTWCDAFCSIRGKRVDLGCAATCPTVKTGVD1QCCSTDDCDPFPTRKRP RCF 76TxC ITP DITSKDCPNGH VCYTKTWCDGFCRIRGERVDLGCAATCPTVKTGVDIQCCSTDDCDPFPTRKRP 77 TxD RCF ITP DITSKDCPNGH VCYTKTWCDGFCSRRGERVDLGCAATCPTVKTGVDIQCCSTDDCDPFPTRKRP RCF 78 TxE KTPIPITSETCAPGQNLCYTKTWCDAWCGSRGKVIELGCAATCPTVESYQDIKCCSTDDCNPHPKQKRP 79 Nno ] TCY VCHTTAT IPSSAVTCPPGENLCYRKMWCDAFCSSRGKVVELGCAATCPSKKPYEEVTCCSTDKCNHPPKRQPG 80 a-B~ . . . .

8! ~c-B~ d 82 Nha 12 83 Nha2a ~NIm3 85 Nha 13b 86 Nhh 11 87 Nhh 2d 88 Nm 4.11 89 Nnv 10 90 Nnk 91 Oh 1 92 Hh lb 93 Hh ICd 94 Da 7' 95 DaB 96 D a 10.2.2 97 D t 13.1A 98 D p p C e 99 D p p 2

I00 Dpp 3 101 D v 4,9.6 '~ ~02 Djk 5.4 103 Djk 5.10 104 Djk 5,1s 101 Djk 4.8 106 Djk 6.4d

+ . . . .

t . . . .

+ - - -

2 . . . .

+ . . . .

3 . . . .

+ . . . .

4 . . . .

+ . . . .

5 . . . .

+ . . . . .

6 . . . . .

+ . . . .

7 . . . .

+ ....

RTCL I S P S S T P Q T C P N G Q D I C F L K A Q C D K F C S I R G P V I E Q G C V A T C P Q F R S N Y R S L L C C T T DNCNH M I C Y K Q R S L Q F F I T T V C P GEKNCYKKQWSG H RGTIIERGC G C P S V K K G I E I N C C T T DKCNR LECY QMSKVVTCKPEETFCYSDVFMP F R N H I V Y T S G C S S Y C R D G T G EK C~TT DRCNGARGG RNH V Y T S G C S S Y C R D G T G EK CCTT DRCNGARGG LECY QMSKVVTCKPEEKFCYSDVFMP F L G K R Y P T G C A A T C P V A K P R E I V E C C S T DRCNH LTCFNCPEVYCNRFHTCRNGEKICFKRFNERKL L G K R Y T R G C A A T C P E A K P R E I V E C C T T DRCNK LTCFICPEKYCNKVHTCRNGENQCFKRFNERKL Q G V E [ K G C V A S C P E F E S K F R Y L L C C R ] DNCNK FTCF TTPSDTSETCPDGQNICYEKRWNS H L G K R Y P R G C A A T C P E A K P R E I V E C C S T DKCNH LTCLICPEKYCNKVHTCRNGENICFKRFYEGNL L G K R Y T R G C A V T C P V A K P R E I V E C C S T DGCNR LRCLNCPEVFCRNFHTCRNGEKICFKRFDQRKL LSR Y I R G C A D T C P V G Y P K E M ] E C C S T DKCNR LRCLNCPEMFCGKFQICRNGEKICFKKLHQRRP DGHVKIERGC GCPRVNPP |SI C C K ] DKCNN L I C F N Q E T Y R P E T T T T C P D G E N CYSTFWHN P N H P V Y L S G C T F C R T D E S G EP CCTT DRCNK LECT QKSKVVTCQPEQKFCYSDT MTFF G M Q I E K G C V A S C P S F E S H Y K F L L C C R I ENCNQ FTCF TTPSDTSETCPIGNNICYEKRWSG H PKMVLGRGC GCPPGDDN LEVKCCTSPDKCNY TMCYSHTTTSRAILTNC GENSCYRKSRRH P PAVVAGRGC G C P S K E M L V A I H C C R S DKCNE MICYSHKTPQPSATITC EEKTCYKKSVRK L PDYISNRGC GCPTAMWP YQTACCKG DRCNK RICYSHKASLPRATKTC VENSCYKMF1RT S P LIIGRGC G C P L T L P F L R I K C C T S DKCN RICYSHKLLQAKTTKTC EENSCYKRSLPK I PKMVLGRGC GCPPGDDY L E V K C C T S P D K C N Y TICYSHTTTSRAILKDC GENSCYRKSRRH P RQYISERGC GCPTAMWP YQTECCKG DRCNK RICYSHKASLPRATKTC VENTCYKMFtRT H K I Y D ] T R G C V A T C P [ P E N Y D S i H C C K T EKCNN LTCVTSKSIFGITTEDCPDGQNLCFKRRHYVVP PGVILARGC G C P K K E 1 F R K S i H C C R S DKCNE MICYSHKTPQNSATITC E E K T C Y K FVTK L PGV]LARGC G C P K K E I F R S I H C C R S DKCNE MICYSHKTPQNSATITC EEKTCYKKFVTN V RGTIIKRGC GCPRVKS K I K C C K S DNCNL RICYNHQSNTPATTKSC VENSCYKS[WAD H TFDN]RRGC G C F T P R G D M P G P Y C C E S DKCNL R I C Y N H L G T K P P T T E CT Q E D S C Y K N I W R N I REYISERGC GCPTAMWP YQTECCKG DRCNK RICYTHKSLQAKTTKSC EGNTCYKMFIRT S STLWWHGCVETCTEDETWKFYRKCCTT NLCNI L E C Y R C G V S G C H L R T T C S A K E K F C A K Q HNR I aMixtureof two homolohom toximwithThr.41 or ile-4LThe sequenceof the majorfractionis shown in the table.bC-tenmnalis Cys-¢9and Cys-60.tone aminoacidresidueis insertedbetweenCys-61and Cys-66,/FromJapanand Philippines.:From the Solomon Japan./Ft~m the Solomon Islands, tQuoted by Karl~on (1973).

K-Bungarotoxitl That Blocks Specifically the Neuronal Nicotinic Acetylcholine Except for (94) a n d (98), Which are Known to be Acetylcholinesterase Toxins. Identical with (33). Speeies

1 2 3 4 5 6 7 8 9

Laticauda colubrina (J.P.)f Laticauda colubrina (S.F.) g Laticauda colubrina (NC) h Laticauda laticaudata (Am)' Laticauda laticaudata (NC) h Laticauda laticaudata ( A m ) i Laticauda laticaudata ( A m ) i Laticauda crockeri Laticauda crockeri 10 Laticauda crockeri 11 Laticauda semifasciata 12 Laticauda semifasciata 13 Laticauda semifasciata 14 Astrotia stokesii 15 Hydrophis cyanocinctus 16 Hydrophis cyanocincigs 17 Enhydrina schistosa 18 Pelamis platurux 19 Enhydrina schistosa 20 Lapemis hardwickff 21 Hydrophis lapemoides 22 Aipysurus laevis 23 Aipysurus laeui~ 24 Aipysurus laevis 25 Pseudechis australis 26 Acanthophi~ antarcticus 27 Dendroaspisjwrae$oni kaimosae 28 Denclroaspis viridis 29 Dendrogspis polylepis polylepis 30 Naja melamoleuca 31 Naja nigricollis 32 Naja n#2ea 33 Naja nivea 34 Naja haje haje 35 Naja haje annulifera 36 Naja haje haje 37 Hemachatus haemachatus 38 Hemachatus haemachatus 39 Naja naja atra 40 Naja naja philippinensis 41 Naja naja oxiana 42 Naja mossambica mossambica 43 Naja mossambica mossambica 44 Naja haje cmnulifera 45 Naja haje armulifera . 46 Laticauda colubrina(Sy 47 Laticauda colubrina(J,P) f 48 Laticauda semtfasciata 49 Astro[ia stokesii 50 Astrotia stokeMi 51 Acanthophis antarcticus 52 Notechis scutatus scutatu~ 53 Dendroaspis viridis 54 Dendroaspis viridis 55 Dendroaspis viridis 56 Dendroaspis viridis 57 Dendroaspis polylepis polylepis 58 Dendroaspts polylepis polylepis 59 Dendroaspis polylepis polylepis 60 Dendroaspgsjamesoni katmosae 61 Ophiophagus hannah 62 Ophiophagus hannah 63 Ophiophagus hannah 64 Naja haje annulifera 65 Naja haje haje 66 Naja melanoleuea 67 Naja melanoleuca 68 Naja haje haje, Desert 69 NaJa nivea 70 Naja naja siamensis 71 Naja naja naja 72 Naja naja naja 73 Naja naja naja 74 Naja naja ( I n d i a n ) 75 Naja naja ( I n d i a n ) 76 Naja naja ( I n d i a n ) 77 Naja naja ( I n d i a n ) 78 Naja naja ( I n d i a n ) 79 Naja na/a oxiana 80 Bungarus multie~ctgs --8

81 Btmgarus multicinctu$ 82 Naja haje annulifvra 83 Naja haje annulifera 84 Naja haje annullfera 85 Naja hoje annullfera 86 Naja haje haje 87 Naja haje haje 88 Naja melanoleuca 89 Naja ntvea 90 Naja naja kaouthla 91 Ophiophagus hanna 92 Hemachatus haemachatus 93 Hemachattts haemachatus 94 Dendroaspis angusticeps 95 Dendroaspis angusticeps 96 Dendroaspis angusticeps 97 Dendroaspis angusticeps 98 Dendroaspis polylepis polylepis 99 Dendroaspis polylepis polylepis 1(30 Dendroaspis polylepis polylepi3 101 Dendroaspis viridis 102 Dendroaspisjamesoni kaimosae 103 Dendroaspisjamesoni kaimosae 104 Dendroaspisjamesoni kaO4nosae 105 Dendroaspisjamesoni kaimosae 106 Dendroa3pisjamesoni kaimoaae

Netwotoxin

Abbreviation

Reference

L a t i c a u d a c o l u b r i n a 1I Latic~uda colubrina c Latlcauda ¢olubrina d L a t i c a u d a laticaudata a L a t i c a u d a laticaudata a L a t i c a u d a latieaudata b L a t i c a u d a laficaudata c L a t i c a u d a erockeri a L a t i c a u d a croekeri b L a t i c a u d a croekeri c Erabutoxin a Erabutoxin b Erabutoxin c Astrotia stokesii a He Hydrophitoxin a He Hydrophitoxin b Es T o x i n 5 Pelamitoxin a Es T o x i n 4 Lh Neurotoxin H y d r o p h i s lapemoides a Aipysurus laevis a Aipysurus laevis b Aipysurus laevis ¢ Pseudeehis australis a A c a n t h o p h i s antaretieus e Djk Toxin Vn-ll D v T o x i n 4.11.3 D p p T o x i n e~ Nm Toxin d N n Toxin ~ N n T o x i n 13 Nn Toxin ~ N h h Toxin CM-6 N h a T o x i n ~t N h h Toxin CM-10a H h Toxin lI Hh Toxin IV Nna Cobrotoxin N n p Toxin N n o N e u r o t o x i n I1 Nmm Seurotoxin I Nmm Neurotoxin IIl Nha CM-10 Nha CM-14 Laticauda eolubrina a Laticauda ooluhrina b L a t i c a u d a semifasciata 111 A s t r o t i a stokesii b A s t r o t i a stokesii c A c a n t h o p h i s anlarcticus b Nss T o x i n 111-4 Dv Toxin I Dv T o x i n V Dv T o x i n 4.9.3 Dv T o x i n 4.7.3 D p p T o x i n Vn2 Dpp Toxin 7/Vnl Dpp Toxin 6 Djk Toxin V n - l l I l Oh Toxin a Oh T o x i n b Oh Toxin CM-9 Nha Toxin III N h h Toxin CM-5 Nm Toxin b N m T o x i n 3.9.4 N h h Desert T o x i n V I I Nn Toxin a N n s 3/ ~ - C o b r a t o x i n N n n Toxin 3 N n n Toxin 4 N n n Toxin 3WP Nn Toxin A Nn Toxin B Nn Toxin C Nn Toxin D Nn Toxin E Nno Neurotoxin I ~-Bungarotoxin

L ¢ 11 Lc c Lcd LI a ( A m ) LI a ( N C ) LI b (Am) LI ¢ ( A m ) l~r a Lcr b Lcr e Ea Eb Ec As a He a He b Es 5 Fix a Es 4 Lh HI a AI a AI b AI c Pa a Aa e Djk Vn-II D v 4.11.3 D p p 0t Nm d Tx a Nnv Nnv 8 Nhh 6 N h a ct N h h 10a Hh II Hh IV Cbt Nnp / q n o I1 Nmm 1 Nmm Ill N h a 10 N h a 14 I.~ a Le b Ls 111 As b As ¢ Aa b Nss 1114 Dv I Dv V D v 4.9.3 D v 4.7.3 Dpp Vn2 Dpp 7 Dpp 5 Djk V n - I l l l Oh a Oh h Oh 9 Nha llI Nhh 5 Nm b N m 3.9,4 Nhh D-VII Nnv ~-Cbt Nnn 3 Nnn 4 Nnn 3WP Tx A Tx B Tx C Tx D Tx E Nno I et-Bgt

T a m i y a et al., 1983b T a m i y a et aL, 1983b T a m i y a et al., 1983b T a m i y a et al., 1983b T a m i y a , N. et aL, unpublished T a m i y a et al., 1983b T a m i y a , N. et aL, u n p u b l i s h e d T a m i y a et al., 1983b T a m i y a et al., 1983b T a m i y a , N. et al., u n p u b l i s h e d S a t o a n d T a m i y a , 1971 Sato a n d T a m i y a , 1971 T a m i y a a n d Abe, 1972 M a e d a a n d T a m i y a , 1978 L i u a n d Blackwell, 1974 L i n a n d Blaekwell, 1974 F r y k l u n d et al., 1972 W a n g ei aL, 1976 F r y k l u n d et aL, 1972 F o x et al., 1977 T a m i y a et aL, 1983a M a e d a a n d T a m i y a , 1976 M a e d a a n d T a m i y a , 1976 M a e d a a n d T a m i y a , 1976 Takasaki, C. a n d T a m i y a , N., u n p u b l i s h e d K i m a n d T a m i y a , 1981b S t r y d o m , 1973a B a n k s et al., 1974 S t r y d o m , 1972 Botes, 1972 L a k e r a n d P o r a t h , 1967 Botes, 1971 Bores et al., 1971 J o u b e r t a n d Taljaard, 1978a Bores a n d S t r y d o m , 1969 J o u b e r t a n d T a l j a a r d , 1978a S t r y d o m a n d Bores, 1971 S t r y d o m a n d Botes, 1971 Y a n g et aL, 1969 H a u e r t et aL, 1974 G r i s h i n et al., 1973 G r e g o i r e a n d R o c h a t , 1977 G r e g o i r e a n d R o c h a t , 1977 J o u b e r t , 1975a J o u b e r t , 1975a K i m a n d Tamiya. 1982 K i m a n d T a m i y a , 1982 M a e d a a n d T a m i y a , 1974 M a e d a a n d Tarniya, 1978 Ma©da a n d T a m i y a , 1978 K i m a n d T a m i y a , 1981a H a l p e r t et aL, 1979 Bechis et aL, 1976 Bechis et aL, 1976 B a n k s et aL, 1974 B a n k s et aL, 1974 S t r y d o m a n d Haylett, 1977 S t r y d o m , 1972 S t r y d o m , 1973b S t r y d o m , 1973a J o u b e r t , 1973 J o u b e r t , 1973 N a n - Q i n et al., 1984 K o p e y a n et aL, 1975 J o u b e r t a n d T a l j a a r d , 1978a Botes, 1972 Shipolini et aL, 1974 S h i m a z u , T. et aL, u n p u b l i s h e d Bores, 1971 K a r l s s o n et aL, 1972 A m b e r 8 et al., u n p u b l i s h e d k A r n b e r g et al., u n p u b l i s h e d ~ R y d d n et al., 1973 N a k a i et aL, 1971 O h t a et aL, 1976 O h t a et aL, 1981a O h t a et al., 1981b H a y a s h i , K , , private c o m m u n . G r i s h i n vtal.. 1974 M e b s et aL, 1972

l¢-Bungarotoxin Nha Toxin CM-12 N h a Toxin CM-2a Nha Toxin CM-3 Nha Toxin CM-13b Nhh Toxin CM-II N h h Toxin CM-2 N m $4CII Nnv Toxin CM-10 N n k Toxin CM-9a Oh DE-I Hh Toxin CM-Ib H h Toxin C M - I C D a T o x i n F7 D a T o x i n Us D a CIoS2C2 D a CI3SICI Dpp Toxin C Dpp FS 2 Dpp Toxin CM-3 D v T o x i n 4.9.6 D j k $5C4 D j k SsC10 D j k $5CI D j k S4Cs D j k $6C4

a m i d a t e d . ¢Trp-29 is oxidized, d o n e a m i n o acid residue is i n s e r ~ d between Islands a n d Fiji. hF r o m N e w Caledonia. F r o m A m a m i Island, K a g o s h i m a ,

~-Bgt N h a 12 N h a 2a Nha 3 N h a 13b Nhh lI Nhh 2 Nrfl 4.11 N n v 10 N n k 9a Oh 1 Hh lb Hh IC Da 7 Da 8 D a 10.2.2 D a 13.1.1 Dpp C Dpp 2 Dpp 3 Dv 4.9.6 Djk 5.4 D J K 5.10 D j k 5A Djk 4.8 D j k 6.4

G r a n t a n d Chiappinelli, 1985 J o u b e r t , 1975a Joltbert. 1977a J o u b e r t , 1977a J o u b e r t , 1975h J o u b e r t a n d Taljaard, 1978b J o u b e r t a n d Taljaard, 1978b Carlsson. 1975 J o u b e r t a n d Taljaard, 1980e J o u b e r t a n d Taljaard, 1980c J o u b e r t , 1977b J o u b e r t a n d T a l j a a r d , 1980d J o u b e r t a n d Takjaard,, 1979b ViUoen a n d Botes, 1973 Viljoen a n d Bores, 1974 J o u b e r t a n d T a l j a a r d , 1980a J o u b e r t a n d Taljaard. 1980a J o u b e r t et al., 1978 S t r y d o m , 1977 J o u b e r t , 1985 Shipolini a n d Banks, 1974 Joubert et al., 1978 J o u b e r t a n d Taljaard, 1979a J o u b e r t a n d Taljaard et aL, 1979a J o u b e r t a n d T a l j a a r d et al., 1980b J o u b e r t a n d T a l j a a r d et al., 1979c

Structure-function relationship of postsynaptic neurotoxins

407

2.2..INvARIANT RESIDUES I n a d d i t i o n to n e u r o t o x i n s , s n a k e v e n o m s f r o m c o b r a s usually c o n t a i n a n o t h e r g r o u p o f small basic p r o t e i n s (60 to 63 a m i n o acid residues) called c a r d i o t o x i n s a n d others. C a r d i o t o x i n s h a v e little affinity for nicotinic A C h R b u t act o n cell m e m b r a n e s c a u s i n g persistent d e p o l a r i z a t i o n (see C h a n g , 1979). D e s p i t e their different m o d e s o f p h a r m a c o l o g i c a l action, n e u r o t o x i n s a n d c a r d i o t o x i n s share a high degree o f h o m o l o g y in their sequences, a n d there are m a n y i n v a r i a n t o r t y p e / i n v a r i a n t residues c o n s e r v e d in b o t h n e u r o t o x i n s

•

4g ~ g~

~g

~

~g~ ~

FIG. 1. Stereo view of the main chain folding (Ca positions) of erabutoxin b at 0.14 nm resolution (Bourne et al., 1985). The side chains of invariant or typc-invariant residues for short neurotoxins (Table 3) are also shown with double lines. The plots were generated by a mol~ular graphics program STDRAW kindly provided by Y. Mitsui (the University of Tokyo), and the coordinates were obtained from the Protein Data Bank. The side chains of most of the conserved functionally important residues are directed away from the viewer on the concave surfac~ of the flat molecule. The insert shows the pattern of the main chain folding and the nomenclature of the three loops.

FIG. 2. Stereo view of the main chain folding (C~ positions) of ~-cobratoxin at 0.28 nm resolution (Walkinshaw et al., 1980). The side chains of invariant or type-invariant residues for long neurotoxins (Table 3) are also shown with double lines. Details as given in FIG, 1.

VP l+ L+

0.30

1.5

N m 3.9.4

Nnv ~ Nno ! ~-Bgt K-Bgt a

69 79 80 81

0.56 0.15

0.08

0.12

Oh a Oh 9

DvV Dv 4.9.3 Dv 4.7.3 c Dpp Vn2 Dpp ? Dpp 8

0.85 0.096 0.098 0.13 0.045 0.08 0.16 0.90 0.4

0.12 0.12

0.05 5.0

0.08 0.08 0.065 0.04

54 55 56 57 58 59 61 63 67

48 Ls IIIb 49Asb 50 As c 51 A a b 53 Dv 1

Long 46Lca 47Lcb

42 N m m l 43 N m m 111 44 Nha l0

39 C b t

26 Aa c

23 Al b

Short 14 As a

Exceptional Ntx

K

+

++ ++ ++ ++ ++ ++ ++ ++ ++ ++ +N +N +N ++ ++ ++ ++ ++ ++ +L

+ + + + + +

+ + + + ÷ + + + + + + + I

++ ++ ++ ++ ++ +A +L + + + + + + + + + + + + + + + + + + + +

t +

G + +

+

G +

2

+ + + + + + + + + + + + + + + + + +

+ +

+ + + + +

KTCP ++++ ++++

C

Cardiotoxin

+ +

C + +

C + +

Short and Long Ntx

+F +

C + +

C +

T + +

+

CY

SS +T ++

1---

+H

N + +

+ . . . .

Long Ntx

. . . .

C + +

0.13

mouse)

(~g/s of

+ +

G + V + +

E D +

+++ ++F

+++ +++ +++ ++F ++F ++F +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++

++ ++

++ ++ ++ ++ ++

NLCYKM +|+F++ ++++++

CY ++ ++

L++

1 CY V++

CY ++ ++

+

+ +

+ + + + + + E E E E + + + E + + + +

+ +

+ M + + +

K R +

+

K R

K + +

D + +

+++ Q++

+D+ +D+ ++N +++ +++ +++ +++ +++ +++ ~++ +++ +++ +++ +++ ++V +++ +++ +++

+ +

+ + + + +

L +

M

W + +

+++

+++

+ G

+ + + + +

D + +

WCD

W + +

3

++ ++

TG TG ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++

+ +

+ + + + +

H F W

++

W+

FC

H F

-+

+N ++ ++ ++ ++ ++ ++

++ ++ ++ ++ ++ ++ ++

K+ F+

+E

++ ++

++ ++ ++ ++ ++

++ +P

E+ E+ ++ ++ ++ ++ ++

++ ++ ++ ++ ++

++ ++ +N

++ ++ ++ ++ ++ ++ ++

++V++++ +++++++ +++++++ +++++++ ++V++++

+++++++ +++++++ +++++++ +++++++ +++++++

+1 +V ++ ++ ++

++ ++ ++ ++ ++

++

+V

GI

+--

++ ++ ++ ++ ++ ++ ++

SLLVKY +A+I+V +++L++

+++++++ ++++I++

+++++++ +++++++ +++++++ +÷+++++ +++++++

++ ++ ++

++ ++ ++

++ ++ ++

CPK +++ +++

VK AR ++

VK AR ++

VK ++ ++

5 . . . .

CP ++ ++

+++ +++ +++ +++ +++ +++ +++

+++++++ +++++++ +++++++

+÷++ ++++ ++++ ++++

+÷Y ++Y ++Y +++

VKRGCID IR+++AN +++++++

GC ++ ++

++++ ++++ ++++

V I L

E D +

GCP +++ +++

+ . . . .

GCAATCP +++++++ +++++++

ERGC +÷++ ++++ VE LD l+

4--

+++ +++ +++

RG K+ ++

++

R+

KG

+++

++S

RGT

. . . .

lnvariant Residue and ( - ) Means a Deletion of an Amino Acid Residue.

Short N t x

Conserved residues

Ncurotoxin

LD~oa

+ + + L

+ +

+ + + +

+ + + + + + +

L l V

1 V +

+

I

L

SD TE +N

SD TE +N

CN +D ++

++

+D

CN

CN ++ ++

+---

++ ++ ++ ++ ++ ++ ++

+++R+ +÷+++ +++++ ++T++

+++++ +++++

÷+ ++ ÷+ ++

+G ++

+÷ +÷ ++ ++

++ ++ ++ ++ ++ ++ ++

TDKCN +NR++ +++++

+++++ +++÷÷ +++++ +++++

++ ++ ++ ++ ++ ++ ++

CC ++ ++

CC ++ +÷

CCSTD ++÷+÷ +++++

CC ++ ++

6 ....

F

~ + P -

÷ +

+ ÷ + +

Y

H

7

+ ....

8

TABLE 3. lnvariant or Type-invariant Residues Among Short Neurotoxins, Long Neurotoxins and Cardiotoxins, and Exceptional Neurotoxins Without any of the lnvariant Residues for Neurotoxins. ( + ) Indicates Conservation of the

>

>

Z

g

Structure-function relationship of postsynaptic ncurotoxins

409

~s

++++ +++++++ +'+++ ++++++++++ + + + + + + + + + + + + + + + + + + + + + + + + + +~+++++~_++ + ++ --

++++ +, ++,÷ +~T

++ ++ ++~+

++~_++~+

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +~

~ ~';~ +;> ~ + +

I +~

+[" +;~"

+ + + +~i ''~

+ ~O m m m m m c° m + to s~ .-I + ;~ -~ -~ )- ~ ~ '~ I ~ + + + ~ ~ + = Z ~ ~ I ~ + ~ + +m + + ~ X ~ m ~ m ~ m m m +~Q<
88

+m~m +~<~

+ ~ + + + + + + + ~ + + + + + + + + + + + ~ + ~ + + + + + + + + + + + + + + + + + + + + + + + + + + ~ ~ + ~ + +++ ++~ ++++ +~ + + + ~

++++ ++++ +mm~ + ~

+ + 1 m

+ + + ~

+ + ÷ -

+ + ÷ +

+ + ÷ ~

+ + ~ +

+ + + + + + + + + + + + + + + + + + + + + + 1 + + ÷ ÷ + + + + + + Q Q Z Q ~ + ~ < < ~

+ + ÷ m

+ + ÷ +

o

+ + = ~

+ - - I ~ ~ ;> ~ ~ ¢¢ ;> ~ , ~ ~ ;> P, ~ ~ >. >.,;> ;> + ~ . + + .~..~. + + + + + t ~ - e = + ~ < z i ~-+++mtor~ +ZZ J ,-10'~-.1~0Z , ~a~m+Ma.~.+[+ I

+++~.~

+ ~ ,

,

+

+

~

.

~

,

+

~

;~o o

~

~

o

+--~

0 ~, ~. ,~ ,~ ~ 0 ~ ,.~Z [. 0 ~ M i. t~ .~ [- ~ M Z + Z [- ,~

+~[~ZZ+~e~=

+ I +'~;~--~'~--=;);~

+ ~ + + + + + + ~ - ~ + + + ~ + + ~ +

++--=: I + ~ Z ~ O

~

+ + + ~ +r~.r~ + + + + + + + ++r~ + + + + +< + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ~ + + + + ~ Z + + + + + + ~ + + + + ~ +

+ + + + + + + + + + + + ÷ + + + + + + + + + + + +

~

~ ~'~

~ +

~ ~

-~-~ ~

+

÷~

~

-~-~ ~

÷ ~

÷ ~

÷ ÷ +~

+ + ~ + ~ + + - - ~ Z + ~ + ~ ~ + ~ + ~ + ~

~

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88

410

T. Erqoo and N. TAMIYA

\2 ~

27

FIG. 3. Stereo view of the main chain folding (C~ positions) of ~t-bungarotoxin at 0.25 nm resolution (Agard and Stroud, 1982; Stroud, 1982). The side chains of invariant or type-invariant residues for long neurotoxins (Table 3) are also shown with double lines. Details as given in FIG. 1.

and cardiotoxins. Such residues include Tyr-25, Gly-44, Pro-50, Leu/Ile/Val/Tyr-58, Ser/Thr763, Asp/Asn/Glu-64 and Asn/Asp-67 and four disulfide bridges, Cys-3 and Cys-24, Cys-17 and Cys-45, Cys-49 and Cys-60, and Cys-61 and Cys-66. Some of these residues may be important in determining the specific tertiary structures common to the two types of the toxins. In contrast, some of the residues are conserved within the neurotoxin family but not in cardiotoxins. These include Lys-27, Trp-29, Asp-31, His/Phe/Trp-33, Arg/Lys37, Gly-38, Glu/Asp-42, Val/Ala-52, and Lys/Arg-53. They probably contribute directly or indirectly to the neurotoxicity but not to the cardiotoxic action. Although a residue may be bifunctional to produce a "neurotoxic structure", attempts have been made to assign a "structural role" and a "functional role" to each of the conserved residues (Ryd6n et al., 1973; Tamiya, 1980). These topics are discussed further in Sections 3.4 and 4.3 The residues conserved within short neurotoxins or long neurotoxins are shown in Table 3. The side chains of these invariant or type-conserved residues for short and long neurotoxins are indicated on the crystal structures of Eb (Fig. 1), ~t-Cbt (Fig. 2) and u-Bgt (Fig. 3), respectively. The exceptional neurotoxins or neurotoxin homologs that lack some of these invariant residues are also summarized in Table 3 with their lethal toxicity. 3. CONFORMATION 3.1. CONFORMATION IN CRYSTALS

3.1.1. Erabutoxin b (Eb)

The molecular conformation of a neurotoxin in the crystalline state was first determined by X-ray diffraction analyses for a short neurotoxin from Laticauda semifasciata, Eb, in Low's laboratory (Low et al., 1976). Tsernoglou and Petsko (1976) also independently determined the crystal structure of a neurotoxin from Philippine sea snake, neurotoxin a, which was Iater confirmed as being identical to Eb (Tamiya and Takasaki, 1978). After refinement of the electron density map, the conformations of crystalline Eb obtained by the two groups (Kimball et al., 1979; Tsernoglou and Petsko, 1976, 1977; Tsernoglou et al., 1977; Bourne et al., 1985) (Fig. 1) are found to be very similar. -The

Structure-function relationship of postsynaptieneurotoxins

411

whole molecule is like a thin, fiat disk in shape. The polypeptide chain is folded into three large main-chain loops bound by four disulfide bridges close to one end. The first loop consists of residues 4 to 16 involving a fl-turn formed by residues 7 to 10, the second loop consists of residues 25 to 44 with a fl-turn at residues 31 to 38, and the third loop consists of residues 50 to 59 with a//-turn at residues 53 to 57. The two strands in the central loops, residues 24 to 31 and residues 38 to 44, and the strand of residues 56 to 61 in the third loop constitute a twisted, three-stranded anti-parallel fl-sheet structure with, at least, 9 hydrogen bonds between peptide carbonyl oxygens and amide hydrogens. This three stranded fl-sheet structure characterizes the unique conformation of this neurotoxin, and it constrains the side chains of several invariant residues such as Trp-29, Asp-31 and Arg37 to point in the same direction from the concave surface of the molecule. When compared with other globular proteins, the molecule has an open structure which is mostly only one peptide thick, but it still has a core structure around the region where disulfide bridges are clustered. 3.1.2. ~-Cobratoxin (~-Cbt) At present, molecular conformations in the crystalline state are available for two long neurotoxins. The first one to be determined was ~t-Cbt from Naja naja siamensis (Walkinshaw et al., 1980) (Fig. 2). The overall arrangement of the polypeptide chain folding is similar to that found in Eb. The three major main-chain loops, which consist of residues 4 to 16, 25 to 44, and 50 to 59, are knotted by four disulfide bridges. There are obviously three peptide segments which adopt the three stranded anti-parallel//-pleated sheet with 11 backbone hydrogen bondings: the segments of residues 24 to 29, 38 to 44, and 57 to 61. The main difference between the crystal structures of ~-Cbt and Eb concerns the extra tail segment of long neurotoxins and the first main-chain loop. A tail piece of nine residues, 67 to 75, hangs down behind the long central loop and the last four residues of the tail (72 to 75) cannot be followed in continuous electron density, suggesting that this part of the molecule may be statistically disordered or has high thermal mobility. The first loop is shorter by three residues in ~t-Cbt than in Eb, and it swings out, protecting the side section of the central loop and allowing the tail segment to cover the back surface of this loop. The overhang of the C-terminal tail may alter the shape of the back surface, and the change in the relative stedc arrangement of main-chain loops may alter the concavity of the front surface of the long neurotoxin molecule. However, the arrangement of the invariant residues, Trp-29, Asp-31, Arg-37, Lys-53, etc. in the central and the third loops of the long neurotoxins is likely to be free from such perturbation. 3.1.3. ~-Bungarotoxin (~-Bgt) ~-Bgt, a long neurotoxin from Bungarus multicinctus, has been widely used for assaying acetylcholine receptor proteins because of its nearly irreversible binding to the receptor. The crystal structure of ~t-Bgt was solved by Stroud and co-workers (Agard and Stroud, 1982; Stroud, 1982) (Fig. 3). The overall polypeptide-chain folding of at-Bgt is similar to that of ~t-Cbt, but there are two major differences. First, while the C-terminal tail segment of ~-Cbt hangs down over the back of the central loop, the tail of ~t-Bgt is swung to the side of the central loop. This difference may be due to the considerable disorder around this part of the molecules, and does not seem to give further insight into the conformations of these two neurotoxins. The second, more significant conformational difference lies in the//-sheet structures. While, with ~-Cbt, the three-stranded /~-sheet structure is predominant in the central and the third main-chain loops, the major portion of the/~sheet structure is disrupted in ~t-Bgt. This difference in the secondary structure is reflected in the fact that, while/~-sheet structure is stabilized by 11 hydrogen bonds in ~t-Cbt, there are only four interchain hydrogen bonds in ~-Bgt, one in the upper part of the central loop and three between the central and the third loop (Kistler et al., 1982). As a result, the side chain of the invariant Trp-29 does not point to the front concave surface as expected in a

412

T. ENDO and N. TAMIYA

regular r-sheet, but is oriented to the back of the molecule. Kistler et al. (1982) have suggested that this singular conformation of a-Bgt probably means the possibility of a conformational change induced in the toxin-receptor binding process. However, this idea is now argued against by the NMR findings (Endo et al., 1981; Inagaki et al., 1985) that aBgt takes a different conformation in solution from that found in crystal, as described in Section 3.2. 3.2. CONFORMATION IN SOLUTION 3.2.1. Spectroscopic Approaches

The results of X-ray diffraction analyses of neurotoxins, Eb, ~-Cbt and ~-Bgt have greatly enhanced our understanding of the structures of neurotoxin molecules. However, the functions of biological molecules, in particular those including protein-protein interactions as in the present case of a neurotoxin and its target acetylcholine receptor, cannot always be explained in terms of rigid structures. Thus, it is necessary to analyze the conformational flexibility as well as the average structure of neurotoxins in solution. For this purpose, a spectroscopic approach, such as NMR, is useful; there are advantages to NMR studies in neurotoxins, because they are relatively small proteins and numerous homologues exist. Before extensive NMR investigations were carried out on neurotoxins, circular dichroism (CD) and laser Raman studies contributed greatly to the understanding of the solution structures of neurotoxins. CD spectra may be analyzed to delineate the secondary structure of a polypeptide backbone of a protein molecule. The CD bands below 220 nm primarily arise from the transitions associated with the peptide backbone while tyrosine and tryptophan residues produce ellipticity in the 220-300 nm region. The reported CD spectra below 240 nm of both short and long neurotoxins show common features: a CD maximum at around 195 nm, a CD minimum at around 210 to 215 mn, and, in some cases, a CD maximum at around 230 mn, as shown in Fig. 4 (Mrnez et al., 1978; Drake et al., 1980; Dufton and Hider, 1983). The common features of CD spectra of most neurotoxins suggest that the neurotoxins have very similar polypeptide chain folding, which had been predicted by Dufton and Hider (1977), Mrnez et al., (1978), and Fox and Tu (1979). The first two bands correspond to the peptide backbone with r-sheet structures and also r-turns. The intensity of the second CD band at 210 to 215 nm is significantly affected by the change in intensity of the first band at around 195 nm. However, a general tendency is found from the CD pattern that long neurotoxins have r-sheet structures with a more pronounced random coil fraction as compared with short neurotoxins. The origin of the third CD band at around 230 nm, which shows a large variation in intensity among neurotoxins, is not clear, but it is perhaps correlated with the presence of aromatic chromophores or disulfide bridges. Laser Raman scattering spectra give more specific information on both backbone and side chain conformations of protein molecules. One example is the Raman spectrum of Eb (Harada et al., 1976) (Fig. 5) which had been reported before the crystal structure was solved by X-ray diffraction analysis. The Raman spectrum shows that Eb contains a large fraction of r-pleated sheet, three out of the four disulfide bridges take the Gauche-GaucheGauche conformation and the remaining one takes the Gauche-Gauche-Trans conformation with respect to the C0t-C~-S~-Sv-Cfl-C~ linkage, Tyr-25 forms a hydrogen bond with a strong acceptor, and Trp-29 is exposed to the solvent. These results are now found to be consistent with those obtained from X-ray diffraction analyses (Kimball et al., 1979; Bourne et al., 1985). Of the various spectroscopic methods, NMR is inherently the most powerful because of its ability to delineate conformational properties of the molecule at the level of atomic resolution. It provides information on both static and dynamic structures, and on the specific intramolecular interactions of the molecules in solution, information which complements that on the static structure provided by X-ray crystallography.

Structure-function relationship of postsynaptic neurotoxins

~

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413

414

T. ENDOand N. TAMIYA

In the NMR analyses of neurotoxins in solution, the averaged, but specific, structural features can be obtained in various ways. For example, one can determine the pK~ values of each ionizable group by NMR, while X-ray crystallography hardly gives direct information on the charge state of the group, pKa may be determined by analyzing the pH dependence of the chemical shift of the proton resonance which senses the ionization of the individual group, pKa values of ionizable groups may be related to the microenvironments of the structurally or functionally important residues in neurotoxins. Proximity relationships between specific residues within a protein molecule may be found by observing nuclear Overhauser effect (NOE), which is the fractional change in intensity of a resonance when another resonance is irradiated (Noggle and Schirmer, 1971). Thus, in the proton NMR spectra of short neurotoxins, Cbt (Endo et al., 1979) and Eb (Inagaki et al., 1980), and long neurotoxins, Ls III (Inagaki et al., 198 lb), ~t-Cbt (Hider et al., 1982), and ~t-Bgt (Inagaki et al., 1985), side chain interactions have been elucidated by NOE measurements. Regular backbone structures of neurotoxins may be revealed by observing NOE among Co-protons, peptide amide protons and other protons, and thus a r-sheet structure that involves the strand with Tyr-25 was found in solution for long neurotoxins, a-Bgt (Endo et al., 1981; Inagaki et al., 1985), Ls III (Inagaki et al., 1982b), and ct-Cbt (Hider et al., 1982; Bystrov et al., 1983). The results obtained for these various short and long neurotoxins are, for the most part, compatible with the crystal structures of Eb and ~-Cbt. Paramagnetic unpaired electrons produce perturbation in the NMR spectra, that is, large shifts and/or pronounced line broadening of signals (Dwek, 1973). This perturbation carries structural information on the interaction between an unpaired electron and a resonance nucleus. Paramagnetic electrons can be introduced in a protein molecule in a noncovalent manner (paramagnetic ions such as lanthanide ions) or in a covalent manner (spin labels). For a short neurotoxin, Cbt, lanthanide Pr(III) and Gd(III) ions were used to study the structural features with respect to carboxyl groups, because, under the experimental conditions employed, carboxylate groups on the surface of the molecule serve as potential weak binding sites for lanthanides (Endo, 1982). It has been shown that paramagnetic lanthanide ions bind to at least three carboxylate groups of Cbt, namely to Glu-2, Asp-31 and Glu-57, but the binding of lanthanide ions to Glu-42 is interfered with by the hydrogen bond between Tyr-25 and Glu-42. A spin label such as a nitroxyl radical perturbs the resonance line widths of the protons within a distance range up to 1.2-1.5 nm from the radical. For Nno II (Naja naja oxiana), monospin-labeled derivatives were obtained for N-terminal, His-33, Glu-2, Lys-15, Lys26, Lys-27, Lys-51 and Lys-53, and furthermore 10 dispin-labeled derivatives were prepared for all the possible pairs of 5 lysine residues (Bystrov et al., 1983). Line broadening of proton NMR resonances and dipolar~lipolar interactions in ESR spectra were analyzed for monospin-labeled toxins and dispin-labeled toxins, respectively, and apparent molecular topology measured from paramagnetic centers were obtained. Similarly, the simultaneously fluorine- and monospin-labeled toxins were prepared and the paramagnetic perturbation of the fluorine NMR spectra was also studied (Arseniev et al., 1981b). Thus, spin-label studies of neurotoxins can complement the NOE results in that it reveals the interaction between the spin label and observed spins which are separated by a larger distance than in the case of NOE measurements (~< 0.4-0.5 nm). 3.2.2. Differences between the Conformations of Neurotox&s & Crystals and & Solution As described in the previous section, CD, laser Raman, and in particular NMR studies on the conformation of various short and long neurotoxins in solution have confirmed that their average structures may be approximated by the common structural features found in the crystal structures of the three neurotoxins. However, the extreme conditions sometimes used for the crystallization of neurotoxins, such as the acidic pH and the presence of heavy metals, together with the effect of molecular packing may induce some

Structure-functionrelationshipof postsynapticneurotoxins

415

conformational changes. Indeed, it has been revealed that neurotoxin molecules also exhibit conformational properties which come out only in aqueous media, but not in crystals. 3.2.2.1. Erabutoxin b (Eb). The molecular conformations of Eb determined in crystal (Tsernoglou and Petsko, 1976; Kimball et al., 1979) contained one point which was apparently inconsistent with the previous results of chemical modification studies. This is on the state of the side chain of one of the two histidine residues in Eb, His-7 (that is corrected to be His-6 as described below). His-7 is hardly iodinated while the other histidine residue, His-26, is readily iodinated under mild conditions (Sato and Tamiya, 1970). On the other hand, solvent accessibility calculations based on the crystal structure of Eb shows that the His-7 side chain is even more accessible to the solvent than the side chain of His-26 (Endo, T., unpublished, results). The state of His-7 in solution, which is not compatible with that in crystal, was also indicated by the NMR analyses of Eb (Inagaki et al., 1978). When the pH of the solution was lowered from the neutral region, the C-2 proton and C-4 proton resonances of His-7 showed little titration shift until denaturation of the whole protein occurred below pH 3.3. This means that His-7 is not protonated at pH > 3.3, whereas histidine residues exposed to the solvent are expected to titrate at pH 6.65 (Tanokura et al., 1978). Besides, the His-7 C-2 proton resonance is unusually shifted upfield, with a secondary shift as large as 1 ppm. Such large shielding effects on a histidine residue suggest a close proximity of another aromatic ring to His-7, but this is not expected from the X-ray diffraction results. All these observations imply that the side chain of His-7 is buried in the protein matrix in the solution state. Inagaki et al., undertook a further detailed NMR investigation on the local conformation around His-7 in solution (Inagaki et al., 1981a). They found by NOE measurement that the imidazole ring of His-7 is close to the Phe-4 aromatic ring but is far from the methyl groups of Ile-40. In crystals, His-7 is near Ile-40 but is far from Phe-4. Clearly, these NMR results are not compatible with the molecular structure in the crystalline state. The conflict of Eb drew much attention of both NMR spectroscopists and X-ray crystallographers because it appeared to provide clear evidence that the conformation of a protein in solution does not always coincide with that in the crystal state. Recently, however, errors were found in the original amino acid sequences of erabutoxins: Gin-6, His-7, Pro-18 and Ser-19 in the original sequence should be His-6, Gln-7, Ser18 and Pro-19. This was discovered by X-ray analyses on the basis of the refined electron density map at 0.14 nm (Bourne et al., 1985), by chemical sequence analyses (Nishida et al., 1985; Bourne et al., 1985) and by cloning and sequencing of cDNA of erabutoxins (Tamiya et al., 1985). Then it was proved that the apparent discrepancy in the conformation of Eb in crystals and in solution was ascribed to the misinterpretation of the X-ray diffraction data. In the early X-ray diffraction analyses of Eb at 0.25 nm resolution, the amino acid sequence before correction had been fitted on the electron density map and the positions of side chain groups of residues 6 and 7 had been found on the basis of the wrong sequence. By tracing the side chains on the density map at 0.14 nm resolution using the corrected sequence data (Bourne et al., 1985), His-6 has now been found to be inaccessible to the solvent; it is surrounded and rigidly held by residues 4, 8, 10, 11, 12, 30 and 39. Besides, the side chains of Phe-4 and His-6 are now adjacent on the same side of the flsheet strand. Thus, the altered three-dimensional structure Of Eb determined in the crystalline state is quite consistent with the interpretation of chemical and NMR solution studies. 3.2.2.2. ~-Cobratoxin (~-Cbt). Since X-ray diffraction analyses of ~-Cbt were carried out on crystals grown in aqueous solution|under acidic conditions, at pH 2.8 (Walkinshaw et al., 1980), it is important to examine if there is any change in conformation of ~-Cbt between acidic pH and neutral pH. Although an NOE contains information on rather short range (~<0.4-0.5 nm) interactions, accumulation of a sufficient amount of NOE data

z

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3

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6

7

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FIG. 6. (a) The N O E diagonal map for ~-Cbt in solution deduced by NMR, and (b) the distance diagonal map for ~-Cbt in crystalline state. In (a), a position (x,y) is marked with (~q), (IZI), or (FIq), when NOE is observed between the amide proton of residue x and the amide proton, the C~-proton, or Ca-proton of residue y, respectively. NOE data by Kondakov et al. (1984a) was used (pH 7.5, 32°C, mixing times for NOESY: 100 ms or 200 ms). In (b), a position (x,y) is marked with (1~), ([]) or ([I]), when the amide proton o f residue x is within the distance o f 0.45 nm from the amide proton of residue y, the C,-proton of residue y, or C~-proton of residue y, respectively. The proton coordinates were generated from the coordinates in the Protein Data Bank (Walkinshaw et al., 1980).

°%

(a)

Z

o~

Structure-function relationship of postsynaptic neurotoxins

417

enables us to delineate the geometrical arrangements of the residues distributed over the whole molecule (Wiithrich et al., 1982). For such systematic analyses of a large data set of NOEs, Bystrov and co-workers have accomplished individual resonance assignments for 67 out of 71 residues of g-Cbt (Kondakov et al., 1984a). On the basis of these assignments, they measured a number of NOEs between distinct protons of the molecules. Fig. 6 is a diagonal map representing the observed NOEs between amide protons of one residue and amide protons, C~-protons and Ca-protons of another residue in ~-Cbt at pH 7.5. This diagonal map may be compared with that obtained on the basis of the atomic coordinates of ~-Cbt in crystal (Walkinshaw et al., 1980). In Fig. 6, a position (x,y) is marked with a square when the amide proton of residue x is within the distance of 0.45 nm from the amide proton, C~-proton(s), or Ca-proton(s ) of residue y. In Fig. 6, one finds that the segments 24-28 and 39-45 and the segments 22-27 and 57-61 form two lines perpendicular to the diagonal, which are characteristic of an anti-parallel E-sheet structure. The short segments 3-8 and 11-16 are also found to be in anti-parallel arrangement. Such secondary structures visualized in the NOE diagonal map (Fig. 6a) are in quite good agreement with the pattern seen in the distance map (Fig. 6b). Thus, overall polypeptide chain foldings of ~-Cbt are shown to be similar in crystal and in solution at neutral pH. On the other hand, a pH-induced local conformational change was found to occur at around pH 5.75 for Tx B (Naja naja), which is very homologous to 0t-Cbt with respect to their sequences; only three residues differ between them. As the pH was lowered from neutral pH to acidic pH, a pH titration shift due to a conformational transition was observed for the proton resonances of Phe-4, His-21, Val-23, Cys-24, Tyr-25 and Leu-43. Most of the other proton resonances, however, hardly shift in this pH region, indicating that the conformational transition is associated with the residues in a very localized region of the Tx B molecule. This local conformational transition has been further confirmed by measuring the pH dependences of the CD spectra (Endo et al., 1982). The backbone CD band exhibited an appreciable pH-induced change with a transition midpoint of 5.7, suggesting that the flsheet structure is lost to some extent in the low pH region. The Hill coetfieient for the CD spectral transition was close to unity and therefore the conformational change in the backbone has little or no cooperativity. Accordingly, the conformational change is induced by the protonation of a single residue, most likely of His-21. Similar conformational transitions triggered by the (de)protonation of His-21 were detected for two long neurotoxins, Nnn 3 (Naja naja naja) (Kondakov et al., 1984b) and, in fact, ~t-Cbt (Hider et al., 1982; Kondakov et al., 1984a,b). Analysis of NOE between distinct protons in the molecule allowed the detailed comparison of 0t-Cbt conformations at two different pH values (Kondakov et al., 1984a). In the conformational state favored at the neutral pH, the deprotonated imidazole ring of His-21 was found to be surrounded by the side chains of Cys-17, Pro-18, Val-23, Cys-24, Cys-45, Ala-46 and Thr-48 residues. In contrast, the protonated imidazole ring of His-21, in the conformational state favored at the acidic pH, is exposed to the solvent. Accordingly, the conformation of 0t-Cbt determined in the crystal may be different from the conformation in aqueous solution at neutral pH, in particular with respect to the local conformation around His-21. This must be always kept in mind when one discusses the structure-function relationship of long neurotoxins on the basis of the crystal structure of 0t-Cbt. Furthermore, the analyses of proton N M R spectra of Tx B on the basis of microscopic equilibrium for the two conformational states of the molecule suggests that the minor conformation, which is favored in the acidic pH region, exists even in the neutral pH region in as much as 10% of the total population (Endo et al., 1982). The interconversion between the two conformations at physiological pH may well influence the biological function of Tx B (and ~t-Cbt).

418

T. ENDO and N. TAMIVA

3.2.2.3. o~-Bungarotoxin (o~-Bgt). According to the crystal structure of 0t-Bgt refined at 0.35 nm resolution (Agard and Stroud, 1982; Stroud, 1982), the global structures were similar to that found for ~-Cbt, but significant differences were found in the region around Trp-29. In 0t-Bgt, the fl-sheet structure around the central loop found in ~-Cbt is lost with some of the associated hydrogen bonds broken, and the invariant residue Trp-29 is oriented in the opposite direction to such conserved residues as Asp-31 and Arg-37. This may be contrasted to the situation of Eb and ~-Cbt, where Trp-29, Asp-31 and Arg-37 are on the same side of the disk-shaped molecule. On the other hand, N M R analyses of ~-Bgt in solution have presented several pieces of evidence that the local structure around Trp-29 is similar to those found in Eb and 0~-Cbt (Endo et al., 1981). The proton N M R spectra of ~-Bgt in 2H20 solution have shown that at least 16 slowly exchanging amide protons can be observed. By combined use of NOE measurement and spin-decoupling experiments, six of the most slowly exchanging amide protons of ~t-Bgt were found to constitute a fl-sheet structure, which probably extends from Cys-24 to Lys-27. These assignments of interior amide protons were confirmed later by using two-dimensional N M R spectroscopy (Inagaki et al., 1985). The existence of such a fl-sheet structure restricts the orientation of the Trp-29 side chain to the same side as that of Tyr-25, Asp-31 and Arg-37. This geometry of the side chains of the residues in the fl-sheet structure was supported also by the observation of NOE between Trp-29 and Val40 (Inagaki et al., 1985). The side chain interaction of Trp-29 and Asp-31 in ~-Bgt in solution has also been observed with N M R (Endo et al., unpublished results). The Trp-29 indole N-1 proton resonance of ~-Bgt shifts downfield on the protonation of Asp-31, indicating that, although the side chain of Trp-29 is not buried, the Trp-29 N-1 proton of 0t-Bgt makes a hydrogen bond with an oxygen atom of a carboxylate group in the proximity. Indeed, no appreciable titration shift was observed for the Trp-29 N-1 proton resonance in the case of Nha 10 (Naja haje annulifera) which has Gly-31 instead of Asp-31 and is unable to form a hydrogen bond between residues 29 and 31 (Endo et al., unpublished results). Thus, the N M R results show that Trp-29 is on the same side as Tyr-25, Asp-31 and Arg-37 in 0t-Bgt in solution. It is in sharp contrast to the molecular conformation of 0¢-Bgt in the crystalline state. The possible interactions between Trp-29, Asp-31, Arg-37, etc. of ~-Bgt and corresponding binding sites of the receptor, which are to be described in sections 4.2 and 4.4, may not differ significantly from those for other neurotoxins under physiological conditions in solution. Indeed in the complex of 0t-Bgt ad AChR, Trp-29 was found to be protected from the tryptophan-specific reagent, 2hydroxyl-5-nitrobenzyl bromide, which could label Trp-29 in free ~-Bgt (Fairclough et al., 1983). Thus, when ~t-Bgt forms a complex with AChR, the Trp-29 side chain may be directed toward the concave active surface of the toxin (see Section 4.4) and lie in contact with the receptor. 3.3. DYNAMIC STRUCTURE

It is now accepted that protein molecules are inherently flexible and contain various levels of internal mobility within their structures. Such properties of proteins must be important in the interactions with other molecules, transmission and change of applied mechanical force, and the stability against the change of surrounding conditions (Williams, 1981). The B factor of atomic positions determined by the X-ray analyses provides some information on the dynamic structure. A large B factor indicates the conformational flexibility and/or mobility in the molecule, or it reflects static disorder in the crystals. Large B factors, as compared with the rest of the molecule, were found for segment 50-55 of Eb (Bourne et al., 1985), and for the C-terminal tail region of residues 71-75 and for segment 9-11 in the first loop of ~-Cbt (Walkinshaw et al., 1980), indicating appreciable disorder in these parts of the molecules. For characterizing the dynamic aspects of protein conformations in solution, N M R methods are particularly effective. N M R studies have demonstrated that neurotoxins have

Structure-function relationship of postsynapticneurotoxins

419

relatively rigid and well-ordered structures but at the same time they have some regional flexibility. For example, some regions of neurotoxin molecules exhibit a temperatureinduced conformational change (or a little expansion of the local conformations) below the overall denaturation temperature; this is also observed in CD spectra (Drake et al., 1980; Dufton and Hider, 1983). In Cbt, spectral transition temperatures of individual protons are not identical; Tyr-25 shows a transition at 66°C, Tyr-39 at 73°C, Trp-29 at 74°C, His-33 at 73°C and His-4 at 67°C and 72°C, and most of the methyl protons and C~-protons show a transition below 68°C (Endo, T. et al., unpublished results). These observations indicate that Trp-29, His-33 and Tyr-39, at the tip of the central loop, constitute a structural module which maintains its structure even above the temperature, 68°C, where a global denaturation prevails in most parts of the molecule. It has been suggested from the temperature dependence of the proton chemical shifts that the region around Thr-13 or 14 and His-4 in Cbt (Endo et al., 1979), that around Val-41 and His-68 in ~-Bgt (Endo et al., 1981; Inagaki et al., 1985), and the/3-sheet structure in Cbt, Eb, Ls III (Endo, T. et al., unpublished results), ~-Cbt (Hider et al., 1982) and ~-Bgt (Inagaki et al., 1985) become more mobile at high temperature. Amide hydrogen exchange with the solvent deuterons also gives information on the conformational rigidity of neurotoxins, because the hydrogen-deuterium (H-D) exchange process of the interior labile hydrogen atoms shielded from the solvent is correlated with a dynamic fluctuation of the molecule. Out of 61-70 backbone amide hydrogens in neurotoxins, about 10-15 amide protons in the fl-sheet structure exhibit an extremely slow rate of H - D exchange under physiological conditions (Endo et al., 1981; Inagaki et al., 1982b, 1985; Kondakov et al., 1984a). Therefore, the measurement of H - D exchange kinetics will provide information on dynamic fluctuation especially around the E-sheet structure of a neurotoxin molecule. This fluctuation accompanies the breaking of single or adjacent hydrogen bonds in the E-sheet segment, resulting in the contact of the amide hydrogens with the solvent molecules. The exchange in the E-sheet was found to be slower at the end near the core part than at the end close to the tip and remote from the core in ~tBgt (Endo et al., unpublished results). The H - D exchange of individual amide hydrogens has been followed for various short and long neurotoxins (Lauterwein et al., 1977; Thi6ry et al., 1980; Nabedryk-Viala et al., 1980; Endo et al., 1981, 1986a, unpublished results). The results suggest that structural rigidity varies with neurotoxins, especially among short neurotoxins; Cbt and Eb may fluctuate more frequently or undergo larger amplitude fluctuation than the other short neurotoxins, Tx ~, AI c (Aipysurus laevis) and As a (Astrotia stokesii). The amide hydrogen exchange rates of long neurotoxins, ~t-Bgt, Tx B, Ls III, As b and Aa b (Acanthophis antarcticus), are slow to the extent comparable to those of the short neurotoxins, Tx ~t, AI c and As a, and no long neurotoxin with amide hydrogens exchanging as fast as those of Cbt or Eb is found. N M R relaxation analyses have proved to be effective in studying conformational fluctuations in the time range of 1 x 10 - s - 1 x 10 -~2 s. 13C relaxation times were measured for the 11 assigned methyl groups of Eb (Inagaki et al., 1982c). The data obtained were analyzed using a restricted diffusion model, which describes restricted diffusion in a cone around the axis of single bonds. The results have suggested that there are large segmental motions (amplitude of the restricted rotation > 50°) for Ile-40, Ile-41 and Ile-56, and much more restricted motion (amplitude of the restricted rotation < 30°) for Ile-2 and Val-65. The mobile residues with large amplitude motions are located in the central and the third main-chain loops, indicating that there is conformational flexibility allowing large concerted motions in this part of the molecule. In contrast, the relatively restricted residues, Ile-2 and Val-65, are located in the core part of the molecule. This conclusion is consistent with the interpretation of proton relaxation time measurements for the assigned methyl proton resonances in Eb, that is, large-amplitude internal motion is present around Ile-40, Ile-41 and Thr-51 (Inagaki et al., 1982a). The flexible region and/or mobile parts in the neurotoxin molecules revealed by these N M R analyses are shown, together with the pH sensitive region described in Section 3.2.2.2, in Fig. 7.

T. Er~oo and N. TAldlYA

420

pH (Tx B,

, Eb)

Temp (i

33 Fro. 7. Flexible and/or mobile regions of (a) short neurotoxins and (b) long neurotoxins revealed by temperature dependence of proton chemical shifts (Cbt, cobrotoxin from Naja naja atra; Eb, erabutoxin b from Laticauda seraifasciata; ~t-Cbt, a-cobratoxin from Naja naja siamensis; Ls III, Laticauda semifasciata III from Laticauda semifasciata; and, ~t-Bgt, ct-bungarotoxin from Bungarus multicinctus); pH dependence of proton chemical shifts (Tx B, toxin B from Naja naja; ctcobratoxin from Naja naja siamensis; and Nnn3, Naja naja naja 3 from Naja naja naja) and ~3C relaxation analyses (erabutoxin b from Laticauda semifasciata). For details, see text. 3.4. MAINTENANCE OF TOXIN STRUCTURE

3.4.1. Structurally Important Residues As mentioned in Section 2.2, neurotoxins contain several invariant residues which are conserved not only in neurotoxins but also in cardiotoxins. Such residues should not be involved in direct interaction with the receptor but may well contribute to the proper polypeptide chain foldings. First, four disulfide bridges, which are common to both neurotoxins and cardiotoxins, should effectively constrain the possible polypeptide chain foldings. When the four disulfide linkages of Cbt are reduced, the toxin loses its native structure resulting in the total loss of neurotoxicity, but the native conformation is restored by the reoxidization of the cysteine residues (Yang, 1965; Mrnez et al., 1980a). Invariant Gly-44 and Pro-50 may be responsible for the specific spatial conformations of neurotoxins. Gly-44 is located in the tightly packed region involving Tyr-25 in the crystal structures of Eb and ~-Cbt, and a bulky side chain in this position would disrupt the side-chain arrangement around this region. Pro-50 is in the loop 49-60, and its potential for fl-turn structure may be favored by the turn in this loop. In crystalline Eb, salt links are formed between the N-terminal amino group and a carboxyl group of Glu-64, and between the C-terminal carboxyl group and a guanidyl group of Arg-43 (Tsernoglou and Petsko, 1976; Kimball et al., 1979; Dufton and Hider, 1983). Since residue 64 is always Asp, Asn or Gin in neurotoxins (and in cardiotoxins) (Table 3), a salt link or hydrogen bond involving residue 64 and the N-terminus may be common interaction among neurotoxins. Residue 43 is always Arg in short neurotoxins (and in cardiotoxins), and therefore the salt link between the C-terminus and Arg-43 appears to be a common interaction among short neurotoxins. Indeed, the N-terminal amino group has a rather high pKa value in short neurotoxins, Cbt (Endo et al., 1979) and in Nmm III (Naja mossambica mossambica) (Arseniev et al., 1981a), indicating that the ~tamino group is close to a negatively charged group. The C-terminal carboxyl group has an

Structure-function relationship of postsynapticneurotoxins

421

unusually low pK~ value (<~ 1) in a short neurotoxin, Nmm I (Naja mossambica mossambica) (Lauterwein et al., 1978), indicating that it is in proximity to a positively charged group. These electrostatic interactions may well stabilize the native conformations of neurotoxins. The roles of type-invariant Val/Leu/Ile-41 and Asp/Asn-67 in neurotoxins are not evident. 3.4.2. Role of the Hydrophobic Core The role of Tyr-25, found in most neurotoxins, was controversial. In the long neurotoxins, ~t-Bgt (Endo et al., 1981) and Ls III (Inagaki et al., 1981b), and in various short neurotoxins (Arseniev et al., 1976; Inagaki et al., 1978; Lauterwein et al., 1978; Fung et al., 1979; Endo et al., 1979; Arseniev et al., 1981a). Tyr-25 exhibited an appreciably high pKa and restricted mobility, indicating that its phenol ring is buried and probably hydrogen bonded with the carboxylate side chain of Glu-42. Laser Raman studies of short neurotoxins, Cbt and Eb, and long neurotoxins, ~-Bgt and Ls III, have also suggested that Tyr-25 forms a strong hydrogen bond with an acceptor (Harada et al., 1976; Takamatsu et al., 1980). Indeed, in the crystal structures of Eb, the side chain of Tyr-25 is dose enough to Glu-42 to form a hydrogen bond with its carboxylate group. Nakanishi et al., (1980) have measured the H - D exchange rate of the OH group of Tyr-25 in Eb by stopped-flow fluorescence spectroscopy, and have found that the deuteration of Tyr-25 exhibits a much smaller (5%) rate and requires a greater activation energy than free tyrosine. In Tx B, a long neurotoxin with Asp-42 instead of Glu-42, the pKa of Tyr-25 is 11.4, which is still higher than that of N-acetyltyrosine methylamide (Endo et al., 1981), and the results of Raman study suggested that it is involved in a weak hydrogen bond (Takamatsu et al., 1976). Since Tyr-25 is a highly conserved residue and position 42 is occupied by Glu or Asp in most neurotoxins, the hydrogen bond between Tyr-25 and Glu/Asp-42 seems to be important for holding the neurotoxin molecule in the biologically active conformation. However, this is not always the case, as will be described below. Chemical modifications of Tyr-25 such as nitration and iodination do not give a clear picture. This is probably because of the differences in the sizes of introduced groups between nitration and iodination, and in the sizes of the side chains between Asp-42 and Glu-42. The nitration of Tyr-25 in Cbt (Chang et al., 1971a) and ~t-Bgt (Chen et al., 1982) causes a global conformational change, resulting in the loss of neurotoxicity. However, for two long neurotoxins, Tx B and ~-Cbt, with a pair of Tyr-25 and Asp-42, the nitration of Tyr-25 does not result in much loss of toxicity (Ohta and Hayashi, 1974a; Karlsson and Sundelin, 1976). The iodination of Tyr-25 in Cbt has no effect on neurotoxicity (Huang et al., 1973). Furthermore, three long neurotoxins without Tyr-25 have been found. As b and As c from a true sea snake, Astrotia stokesii, have Phe-25 and Ile-42, and Phe-25 and Val-42, respectively. Aa b from an Australian land snake, Acanthophis antarcticus, has Phe-25 and Glu-42. Although these toxins cannot form a hydrogen bond between residues 25 and 42, they are fully neurotoxic (Table 3). Careful NOE measurements for these three neurotoxins revealed that the side chain of Phe-25 is close to those of Ile/Val-42 (As b and As c), Ala-46 and Ile/Val-58 (Endo et al., 1986b). Thus, in long neurotoxins it is the hydrophobic core consisting of residues 25, (42), 46, 58, and probably along with residues 23 and 27 (Inagaki et al., 1981b; Hider et al., 1982) that contributes to maintaining the biologically active polypeptide chain folding. In long neurotoxins, another hydrophobic cluster probably exists. This was suggested by the observation that Tyr-4 in long neurotoxins, As b and Aa b (Endo et al., 1986b), or Phe-4 in another long neurotoxin, Tx B (Endo, T. et al., unpublished results), is close to solvent-inaccessible interior amide protons. Indeed, NOE analyses on long neurotoxins have shown the proximity relations among residues 4, 7, 41, 43 and 69 for Ls III (Inagaki et al., 1981b) and among residues 4, 7, 43 and 69 for ~-Cbt (Hider et al., 1982). In the crystal structure of ~-Cbt, the side chains of hydrophobic residues 4, 7, 41, 43 and 69 are on the concave surface of the disk-shaped molecule, and those of residues 23, 25, 27, 42,

422

T. Er~oo and N. TAMIYA

46 and 58 lie on the convex side of the molecule. The fact that no NOE is observed between Tyr-4 and Phe-25 in As b and Aa b indicates that these two residues are not in close proximity and supports the above mentioned side chain arrangements. Thus, two distinct hydrophobic clusters are likely to be formed around Phe/Tyr-4 and Tyr/Phe-25, and they may make together a large hydrophobic-interaction network in the center of the long neurotoxin molecule where disulfide bridges are clustered. For short neurotoxins, the results of N M R analyses of Eb (Inagaki et al., 1980) and Cbt (Endo et al., 1979) in solution have shown that the aromatic ring of Tyr-25 is close to Ile/Leu-58 and Lys-27. Since short neurotoxins do not have hydrophobic residues in positions 23 or 46, only the side chains of residues 25, 27 and 58 may be incorporated in the hydrophobic clusters around Tyr-25 of short neurotoxins. Short neurotoxins have Phe, Tyr, His or Cys in position 4, and Thr or Ile in position 41, and they have no hydrophobic residues in positions 7, 43 or 69. Therefore, a hydrophobic cluster may not be constructed around residue 4 in short neurotoxins. It is thus clear that long neurotoxins have a larger hydrophobic core than short neurotoxins. With this large hy~trophobic core of long neurotoxins, the hydrogen bond between Tyr-25 and Glu/Asp-42 may not be essential for the maintenance of the native conformation. The stable hydrophobic core primarily contributes to the proper mainchain folding of long neurotoxins, and probably also to the generally enhanced thermal stability of long neurotoxins (Miyazawa et al., 1983; Endo et al., 1986a).

4. INTERACTION WITH THE RECEPTOR 4.1. STRUCTURE OF THE ACETYLCHOLINE RECEPTOR The target of neurotoxins is the nicotinic acetylcholine receptor at the postsynaptic membrane of skeletal muscle or electric organ, which mediates synaptic transmission by translating chemical information into an electrical response. The binding of the neurotransmitter acetylcholine to AChR leads to the receptor ion-channel opening so that the permeability of the synaptic membrane increases to allow the passage of cations. This depolarizes the endplate membrane at the neuromuscular junction, resulting finally in the muscular contraction. Because of its high density of distribution, AChR from fish electric organs of Torpedo, Narcine, and Electrophorus has been extensively studied (see Conti-Tronconi and Raftery, 1982; Changeux et aL, 1984: McCarthy et al., 1986 for reviews). The receptor is a pentamer consisting of four kinds of transmembrane polypeptide subunits with the molar stoichiometry of 0t2flyr. In calf muscle, the ~-subunit may be replaced by the ~-subunit, and this replacement of the 7-subunit by the g-subunit takes place during muscle development (Takai et al., 1985; Mishina et al., 1986). The complete primary structures of these polypeptide chains comprising fish electric organ AChR or mammalian or avian AChR have been deduced by cloning and sequencing the cDNAs in the laboratories of Numa (Noda et aL, 1982, 1983a,b,c; Tanabe et al., 1984: Takai et al., 1984, 1985; Kubo et al., 1985; Shibahara et al., 1985) and others (Sumikawa et al., 1982; Claudio et al., 1983; DevillersThiery et al., 1983, LaPolla et al., 1984; N e f e t al., 1984). Electron microscopy studies have shown that the 5 subunits of AChR are arranged in a dimer of rosette-like structures, both of which probably form gated ion channels in the centre (Zingsheim et al., 1982a,b; Brisson and Unwin, 1985). The channel is like a funnel, which is 0.25-0.30 nm in diameter at the synaptic end of the oligomer and which becomes very narrow at the cytoplasmic end (Ross et al., 1977; Klymkowsky and Stroud, 1979; Kistler et al., 1982). A model of the receptor structure is illustrated in Fig. 8. Acetylcholine binding to AChR in a "resting", or non-liganded state leads to a rapid ion-channel opening, and within a few milliseconds, the channel is closed again at a slower rate. Upon prolonged exposure to cholinergic agonists, AChR undergoes another transition to a state with increased affinity for these lig.ands. This takes place at a much slower •

Structure-function relationship of postsynaptic neurotoxins

423

rate, in the second to minute time scale, and this transition is probably correlated with the onset of functional "desensitization". In the central nervous system or neurons in culture as well as vertebrate skeletal muscle and fish electric organ, high affinity binding sites for postsynaptic neurotoxins have been found, and in some cases, these are probably neuronal nicotinic AChR (reviewed by Oswald and Freeman, 1981, and Morley and Kemp, 1981). ~-Bgt binding site from chick optic lobe displays similar ligand-binding and immunological characteristics to muscle AChR (Norman et al., 1982), and the N-terminal amino acid sequence of its lowest molecular weight component suggests that this AChR is closely related to muscle AChR (Conti-Tronconi et al., 1985). However, different nicotinic AChR that is not sensitive to ~Bgt binding may also exist in the central nervous system because ~-Bgt binding does not always block neuronal synaptic transmission (Oswald and Freeman, 1981; Morley and Kemp, 1981). In neurons in embryonic chicken ganglia, binding sites for antibodies raised against AChR from muscle or electric organ were found not to bind to ~-Bgt (Jacob et al., 1984). The sequence of a cDNA coding for a possible neuronal AChR ~-subunit has been determined recently (Boulter et al., 1986). 4.2. BINDING SITES OF THE ACETYLCHOLINE RECEPTOR FOR CHOLINERGIC LIGANDS AND NEUROTOXINS

Neurotoxins bind to AChR with a stoichiometry of 2:1 and in a strictly competitive manner with cholinergic agonists and antagonists, or with the affinity label MBTA. Binding of a neurotoxin to AChR blocks the binding of acetylcholine but does not induce ionchannel opening, resulting in the block of neurotransmission. Out of the four isolated subunits of AChR, which are separated by electrophoresis and then partially renatured or immobilized into protein blots, only the ~-subunit is capable of binding to ~t-Bgt (Haggerty and Froehner, 1981; Gershoni et al., 1983). Specific binding of a neurotoxin to the ~-subunit of AChR has also been confirmed by observing the effect of the deletion of a-subunit(s) on g-Bgt binding activity of the AChR which was synthesized by the cloned cDNAs encoding three out of the four subunits of the Torpedo californica receptor

~

~."¸i." • ~'~

~ii~~;.";~: ,~ :!~:./

FIG. 8. A model of the molecular structure of AChR in the postsynaptic cell membrane. Five AChR subunits are arranged around the ion channel, but the disposition of the subunits is not conclusively established. Two AChR molecules are linked by a disulfide bridge b~twecn the two t$subunits. In the model by Finer-Moore and Stroud (1984), ~ach subunit contains four hydrophobic (M1, M2, M3 and MS) and one amphipathic (M4) transmembrane helices, but this model was modified to accommodate an additional hydrophobic sequence (M6) and amphipathic helix (MT) (Criado et al., 1985). Recently Ratnam et al. (1986) have proposed a new model in which M4 and M5 arc not in the membrane but on the cytoplasmic surface. M7 likely forms an amphipathic helix which may line the cation channel. Only the amphipathic helices arc drawn. An extraceliular part of the a-subunit on the synaptic side contains an acetylcholine and/or neurotoxin binding site close to Cys-128, Cys-142, Cys-192 or Cys-193 and an N-glycosylation site at Ash-141. Carbohydrate structures of AChR have bc~n determined recently (Nomoto et aL, 1986).

424

T. ENDOand N. TAMIYA

(Mishina et al., 1984). The effect of binding of neurotoxins to the two ~-subunits of the receptor molecule was directly observed by electron microscopy with single-particle image averaging (Zingsheim et al., 1982a). The binding sites on the two ~-subunits do not appear to be equivalent because one of them is more susceptible to affinity labeling after reduction of the disulfide bride close to the binding sites (Wolosin et al., 1980). Two classes of binding sites for agonists have also been reported in the literature (Schiebler et al., 1977; Fels et al., 1982). This nonequivalence could be due to the difference in the extent of N-asparagine glycosylation at residue 141 (Conti-Tronconi et al., 1984) or to the asymmetric quaternary structure of AChR with respect to the two ~t-subunits. The situation seems similar for the antagonist and/or neurotoxin binding sites on the ~-subunits: there is also evidence showing that the two sites are not equivalent, d-Tubocurarine binds to different sites on AChR with different affinities (Neubig and Cohen, 1979). The affinity labeling of the ~-subunits with MBTA decreases linearly as the extent of neurotoxin occupancy of the toxin binding sites increases, suggesting the two toxin binding sites are not identical (Damle and Karlin, 1978). Ellena and McNamee (1980) examined the electron spin resonance (ESR) spectrum of spin-labeled ~-Cbt when bound to the two toxin-binding sites of AChR and when attached to one of the two toxin-binding sites of AChR, where the other toxin-binding site was blocked by MBTA, and concluded that two toxin-binding sites are very similar. On the other hand, Rousselet et al. (1984) have found that a neurotoxin derivative with a spin label at Lys-53, when bound to AChR, yields a complex ESR signal, which suggests a physical difference between the two toxin binding sites on the receptor. If there are two toxin-binding sites with different affinities on an AChR molecule, it may be reflected in the association kinetics of a neurotoxin and the receptor. However, there is no general agreement as to whether toxin-receptor interaction follows a single mechanism or not. Association kinetics of ~t-Bgt with membrane-bound AChR have been found to be monophasic (Blanchard et al., 1979; Lukas et al., 1981), or biphasic (Leprince et al., 1981). Such different results reported in the literature may be due to the design of the experiments (Lukas et al., 1981) or to the methods employed for the preparation of AChRenriched membrane particles (Dunn et al., 1983). Maelicke and co-workers (Maelicke et al., 1977; Kang and Maelicke, 1980) have observed that the association process of ~-Cbt, or fluorescein isothiocyanate-labeled ~t-Cbt, and AChR is monophasic, but that the disspciation of the toxin-receptor complex is composed of two kinetic processes. However, Weiland et al., (1976) have found that both processes are monophasic. In the case of Maelicke et al. (1977), biphasic kinetics observed only in the dissociation process was explained by assuming a conformational transition between the two states of the toxin-receptor complex with different affinity rather than by assuming a non-equivalence of the two toxin-binding sites. Affinity labeling experiments have shown that the two ~-subunits carry binding sites for cholinergic agonists and antagonists within 0.10nm of a reducible disulfide bridge (Karlin, 1980). Mishina et al. (1985) have separately replaced each of the five cysteine residues (Cys-128, Cys-142, Cys-192, Cys-193 and Cys-222) in the putative extracellular part of the ~-subunit by serine residues with the aid of site-specific mutagenesis of the cloned cDNAs of Torpedo californica AChR. Using the expression system of Xenopus oocytes, they examined the effects on the amount of synthesized AChR polypeptides, the binding ability for ~-Bgt and the ability to respond to acetylcholine. The AChR mutants with (Cys--,Ser)-192 or (Cys--,Ser)-193 have nearly normal ~-Bgt binding activity but the mutants show a decreased affinity for the agonist and no response to acetylcholine at all. The substitution of Cys-128 or Cys-142 with serine residues decreased the synthesis of the polypeptide, and the product showed almost no ~-Bgt binding ability and failed to respond to acetylcholine. Thus, the authors have suggested that Cys-192 and Cys-193 of the at-subunit play specific roles in acetylcholine binding and in subsequent signal transduction into the ion-channel opening, whereas Cys-128 and Cys-142 are essential for maintaining the proper folding or assembling of the AChR subunits. It should be noted, however, that when a cysteine residue is converted to a serine residue, the remaining

Structure-function relationship of postsynapticneurotoxins

425

disulfide-bridge partner may perhaps replace one of the other two Cys in the formation of a disulfide bridge, resulting in a severely altered binding site (White, 1985). The identification of Cys-192 and Cys-193 as a binding site for acetylcholine is consistent with the findings that 3H-labeled 4-(N-maleimidobenzyl)trimethylammonium (MBTA), an affinity label specific to the location near the AChR binding site, is introduced to Cys-192 (Kao et al., 1984). On the other hand, Cahill and Schmidt (1984) have found, by using monospecific anti-MBTA antibodies, that MBTA covalently binds to the site near Cys-142. The segments around Cys-128 and Cys-142, and around Cys-192 and Cys-193 bear candidates for possible binding sites for acetylcholine or neurotoxins. Model-building studies have shown that residues 135-138 in the ~-chain of Torpedo californica AChR favor B-turn conformation and that Asp-138 and Cys-142 are positioned on the same side of the B-sheet structure separated by a distance of 1.0 nm (Smart et al., 1984). In this model, Asp-138 may be an acceptor site for the quaternary ammonium ion of acetylcholine and Lys-53 of a neurotoxin, and Phe-137, Gin-139 and Gin-140 in the AChR ~t-chain may be interacting partners of neurotoxin residues Trp-29, Thr-51 and Glu/Asp-42, respectively. Gin-140 of the ~-subunit chain may provide a hydrogen-bond donor for the carbonyl oxygen of acetylcholine (White, 1985). In the re#on around Cys-192 and Cys193, there are several negatively charged residues, Asp-180, Asp-195 and Asp-200. One of these residues may well represent the anionic subsite for the acetylcholine binding. As described above, the binding region for a neurotoxin within the ~-subunit of AChR has been searched for on the basis of the established amino acid sequences of the AChR ~subunit chain. Mutually exclusive competition of the binding of a neurotoxin and a cholinergic agonist to AChR strongly suggests that the binding sites for neurotoxin and acetylcholine overlap, at least in part, on the receptor. However, single amino-acid substitution of Cys-192 or Cys-193 on the ~t-subunit reduces the ability to bind acetylcholine without altering the ability to bind neurotoxins, indicating that the binding sites for a neurotoxin and acetylcholine are not identical (Mishina et al., 1985). Neumann et al. (1985) have prepared antibodies to the segments comprising residues 1-20 and residues 126-143 (including Cys-128 and Cys-142), and tested binding of these antibodies to various proteolytic fragments of the ~-subunit of Torpedo AChR. The fragment, which could bind to ~-Bgt, was found to react with neither of the antipeptide antibodies. Therefore, the toxin-binding site may not lie in proximity to Cys-128 and Cys-142 on the ~-chain sequence. Further studies on those proteolytic fragments of the ~-subunit have shown that the toxin-binding fragment initiates beyond Asp-152 and terminates in the region of Arg313/Lys-314 (Neumann et al., 1986). Wilson et al. (1984) have obtained proteolytic fragments of the ,t-subunit of Torpedo californica AChR, some of which carry binding sites for both ~-Bgt and the affinity label, MBTA, although the neurotoxin binds to the fragments with reduced affinity. These proteolytic fragments that bind to ~-Bgt have been analyzed on the basis of the size of the fragments and the presence of Asp-141 as determined by susceptibility to digestion with endoglycosidase H (Wilson et al., 1985). It was revealed that the ~t-Bgt binding site is present within the ~t-chain segment 153-241, and further that a synthetic peptide comprising residues 173-204 of the ~-subunit of the Torpedo AChR binds to ~t-Bgt. The shorter synthetic peptide corresponding to residues 185-196 of the ~tsubunit of Torpedo AChR was also shown to bind to ~-Bgt and this binding was inhibited by competition with d-tubocurarine (Neumann et al., 1986). Criado et al., (1986) prepared monoclonal antibodies to the synthetic peptide corresponding to the ~t-chain segment 127-143 of the Torpedo AChR and showed that cholinergic ligands had no or little effect on binding to the receptor of the antibodies. In addition, they also found that the monoclonal antibodies, when bound to the receptor, did not affect toxin-bindin_g kinetics. Thus, the authors concluded that segment 127-143 of the ~subunit of AChR is probably not involved in the binding site for cholinergic agonists and antagonists. Atassi and co-workers synthesized peptides containing two possible binding regions of the ~-subunit of AChR, which involve Cys-128 and Cys-142, and Cys-192 and Cys-193 respectively (McCormick and Atassi, 1984; Mulac-Jericevic and Atassi, 1986). These

426

T. ENooand N. TAM~YA

peptides consist of the region 182-198 and the region 125-147 of the or-chain of Torpedo californica AChR and the region 125-148 of the ~t-chain of human AChR. Interestingly, all the peptides exhibited high affinity for ~t-Bgt or ~-Cbt. Furthermore, binding of toxins to the peptide 182-198 was inhibted by the Torpedo californica peptide 125-147 as well as by the human peptide 125-148. These results, in contrast to the observations described above, suggest the involvement of both of the two regions in the neurotoxin binding site(s) of the receptor. That is, the ~t-subunit of AChR contains two toxin binding regions and they constitute a single larger binding site for the toxin, or they are spatially so close that the binding of one synthetic region to the toxin sterically interferes with the binding of the second synthetic region. As already described, ~-Bgt and most neurotoxins from cobras and sea kraits bind to the two ~-subunits of AChR with the stoichiometry of 2 : 1. However, Conti-Tronconi and Raftery (1986) have examined the stoichiometry of the binding of a neurotoxin from a mamba, Dv 4.9.3 (Dendroaspis viridis), to AChR, and found that 4 moles of toxin bind to 1 mole of AChR, although ~-Cbt or ~t-Bgt gives a stoichiometry of 2:1 for the binding to AChR. They pointed out the possibility that AChR contains two low-affinity binding sites for cholinergic ligands, which are blocked by Dv 4.9.3 but not by ~-Bgt or ct-Cbt, in addition to the high affinity binding sites for cholinergic ligands on the two ~-subunits (Dunn and Raftery, 1982a,b; Dunn et al., 1983). The low affinity binding sites for cholinergic are probably on subunits other than the ~-subunits. When affinity labeling of a neurotoxin was tested on a complex of AChR subunits, cross linking of the neurotoxin was found to occur sometimes not only with the ~t-subunit but also with other subunits (Witzeman et al., 1979; Nathanson and Hall, 1980; Oswald and Changeux, 1982; Hamilton et al., 1985). This is not surprising because the putative binding surface region of a neurotoxin is about 3.0 x 2.0 nm and the receptor subunits are organized in a circle of about 6.0 nm in diameter, so that a neurotoxin may well be in contact with subunits other than the ~t-subunit. In such cases, the results of cross linking contain information on the arrangement of the AChR subunits with respect to one another. Witzemann et al. (1979) prepared photoaffinity derivatives of ~-Bgt with aryl azide side chains of different lengths. A derivative with a 1.4 nm side chain cross-linked to the ~- and 6-subunit chains, while that with a 3.3 nm side chain cross-linked exclusively to the 6-subunit. Oswald and Changeux (1982) showed that 125I-~-Bgt could be cross-linked covalently to the ~-, ~- and 6-chains of the receptor by simple u.v. irradiation. Hamilton et al. (1985) cross-linked 3H-methyl ~-Cbt activated by dithiobis-(succimidyl propionate) to each of the AChR subunits, and found that the cross-linking pattern of each subunit was changed significantly by prior incubation with agonists or antagonists, or by carrying out reduction and alkylation of the receptor. The subunit arrangement of AChR, which was consistent with the change of cross-linking pattern, was ~fl~),6. The correlation between the formation of neurotoxin binding site(s) and the development of functional AChR during biosynthesis is also a matter of interest. Carlin et al. (1986) have identified the AChR precursor, which consists of the ~-subunit alone or with another subunit in embryonic rat myotubes in primary culture. They have found that ~Bgt binds to the precursor ~-subunit but that this binding is not inhibited by the cholinergic ligands, decamethonium, or d-tubocurarine. This suggests that, in the posttranslational processing of AChR, toxin binding properties are first acquired and then, probably by the assembly of the 5 receptor subunits, the d-tubocurarine binding site or cholinergic ligand binding site is developed. In other words, a part of toxin binding sites is formed earlier in the biosynthesis than the rest of the toxin binding sites that are common to cholinergic ligand binding sites. Boulter et al. (1986) have isolated and sequenced a cDNA coding for a possible neuronal AChR ~-subunit of mouse, which hybridizes to RNA species in various neuronal tissues but not to those in muscle tissue. Amino acid sequence of this neuronal AChR ctsubunit shows high homology with that of mouse muscle AChR ~-subunit including Cys128, Cys-142, Cys-192 and Cys-193. However, the sequences surrounding the above four Cys residues are not conserved in neuronal AChR ~t-subunit. There are substitutions at 4

Structure-function relationship of postsynaptie neurotoxins

427

out of 13 residues between Cys-128 and Cys-142 and two of these substitutions are not conservative changes, i.e. Lys-129 for Glu-129 and Asp-130 for Ile-130. Between residues 153 and 172, only one residue out of the 20 residues are conserved and 8 out of 19 substitutions are non-conservative changes. If the latter region in this AChR is involved in cholinergic ligand and/or toxin binding, such sequence differences in this region between neuronal AChR and muscle AChR may account for the reduced affinity of neurotoxins with AChR in the central nervous system. 4.3. FUNCTIONALLYESSENTIALRESIDUESOF NEUROTOXINS 4.3.1. Functionally Essential Residues Common to Short and Long Neurotoxins Ishikawa et al. (1977) have examined the relation between the affinity of neurotoxins to AChR and their lethality as determined by LDs0s in mice. The lethal dose of a neurotoxin reflects its concentration in the target tissue, the respiratory muscles, at which a certain fraction of the receptor is occupied by the toxin (Barnard et al., 1971). Then the following equation is assumed to hold in the target tissue, although the tissue is not homogeneous. ([AChR]0 - [AChR • Ntx])([Ntx]0 - [AChR. Ntx]) = K~t~ [AChR • Ntx]

(1)

where [AChR]0 and [Ntx]0 are the total concentration of the receptor and the toxin, respectively, and [AChR • Ntx] is the concentration of the toxin-receptor eomplexed, all in the target tissue. The apparent dissociation constant K'N,~ is related by a factor f to the dissociation constant KNtx obtained with Torpedo receptor; i.e. K'Ntx= f KNOx-The LDso should be proportional to the concentration [Ntx]0 ([Ntx]0 = flLDs0), with which a certain mole fraction of the receptor is occupied by the toxin and the animal will die with 50% probability. Then, one obtains the equation below, ~

~t

LDs0 - fl (1 - ~ fKNtx + -~ [AChR]0

(2)

Indeed, a linear relationship between LD~0 and KNt~ has been found for neurotoxins with normal toxicity, Ls III, and two chemically modified derivatives of Eb with (~tf)/{fl(1-~)}--65 and ~/fl= 10-a (Ishikawa et al., 1977). Equation (2) implies that if a neurotoxin has very low affinity for AChR, its LDso value will be proportional to KNox.On the other hand, a neurotoxin with sufficiently high affinity (i.e. a small value for KNtO will have an LD~ that is not proportional to KNt~and is close to the constant value (~/[3) [AChR]0, which is the minimal dose of the neurotoxin to kill an animal. This should be kept in mind in the following discussion on the relationship between the chemical modification or amino-acid replacement of neurotoxins and their toxicity. The binding of a neurotoxin to AChR is usually specific and tight (Weber and Changeux, 1974): the dissociation constant of a toxin-receptor complex is in the range of l0 - 9 - l0 -it M while that of a receptor-acetylcholine complex is about three orders of magnitude larger. Such specific recognition and high affinity for the toxin-receptor interaction may well require multi-point interactions between the two proteins. Amino acid residues of neurotoxins responsible for the interaction with the receptor may be deduced by examining the relationship between amino-acid substitution in neurotoxins and their lethal doses. As seen in Table 3, some neurotoxins have higher lethal doses than other neurotoxins, meaning that, in these toxins, amino acid replacements have occurred in critical positions of the sequences and reduced the neurotoxicity. Chemical modification of neurotoxins has also been employed frequently to deduce the functionally essential groups of toxins (reviewed in Karlsson, 1979). It is also important to examine the solvent accessibility of the invariant residues, because the residues involved in the initial recognition step of the binding to the receptor are likely to be exposed. Since cloning of the gene coding for Ea has been accomplished (Tamiya et al., 1985), artificial substitution of amino

428

T. E~oo and N. TAMIYA

acid residues by site-directed mutagenesis would be a promising method for identifying residues responsible for the function, but such an approach has not been undertaken yet. Since AChR is a complex of acidic protein subunits, and since neurotoxins and other low-molecular-weight agonists and antagonists are all cationic, basic molecules, it is natural to assume that cationic groups of neurotoxins interact with the receptor. Selective trinitrophenylation (Chang et al., 1971b), acetylation (Hori and Tamiya, 1976; Tsetlin et al., 1979a; Faure et al., 1983) and biotinylation of Lys-53 (Lobel et al., 1985) increase the lethal dose, reducing the affinity for AChR. Guanidination, which preserves the positive charge, of Lys-53 has a rather small effect on the toxicity (Chang et al., 1971b). Most of the neurotoxins have lysine or arginine residues in position 53 except for Nno I (Naja naja oxiana) with Glu-53 and Ls III with Asn-53, both of which are somewhat weak as neurotoxins (Table 3). Thus, the removal of a positive charge from the highly conserved Lys- or Arg-53 reduces the affinity of the toxin for the receptor significantly. The c-amino group of Lys-53 appears to be exposed in Eb, because it has a normal pKa value (Inagaki et al., 1980). Modification of the nearly conserved Lys-27 gives a different effect on the toxicity for different neurotoxins. Lys-27 acetylated Tx ~t (Faure et al., 1983), Lys-27 biotinylated ~-Cbt (Lobel et al., 1985), Lys-27 acetylated Eb (Hori and Tamiya, 1976) and Lys-27 acetylated Nno II (Tsetlin et al., 1979a) lose their activity, but Lys-27 trinitrophenylated Cbt (Chang et al., 1971b) retains full activity. M6nez and Tamiya (1982) have measured the thermal denaturation temperatures of Eb and its derivatives, monoacylated at a single lysine residue, and have found that the acylation of Lys-27 increases the denaturation temperature by 5°C. The increase of thermal stability by abolition of the positive charge of Lys-27 suggests that, in Eb, the c-amino group of Lys-27 in a hydrophobic environment or in proximity to another positively charged group destabilizes the native state. This is also supported by the rather low pKa value (9.7) of Lys-27 ~-amino group in Eb (Inagaki et al., 1980). Position 27 is occupied by a lysine residue in most neurotoxins, but there are several neurotoxins that have Glu-27 or Met-27 and still have full toxicity (Table 3). Therefore, one possible explanation is that modification of Lys-27 causes some environmental pertubation on other residues in the neighborhood, and this diminishes the toxicity to a certain extent in some cases. It is to be noted that Dv 4.9.3, which binds to AChR with a molar stoichiometry of 4:1, has a glutamic acid in position 27. An arginine residue with a positive charge is conserved in position 37 without exception. Modification of Arg-37 in Cbt reduces the toxicity significantly (Yang et al., 1974). Therefore, Arg-37 should play an essential role in neurotoxicity. Apart from lysine and arginine residues with positive charges, other conserved residues (shown in Table 3) may also be involved in neurotoxicity. For example, Asp-31, which is probably hydrogen bonded with Trp-29, is preserved in most neurotoxins except for Nha 10 and Nha 12 (Naja haje annulifera), which have weak neurotoxicity (Table 3). The competition binding experiments of Nha 10 or Nha 12 with AChR in the presence of 3Hlabeled Tx ~t has revealed that Nha 10 with Gly-31 instead of Asp-31 still has 20% AChR binding activity of normal neurotoxin, Eb (Endo, T. et al., unpublished results), and is only five times less potent than Nha-14 at blocking neuromuscular transmission in vitro (Harvey et al., 1984). Thus, Asp-31 plays a part in AChR binding, but it is not essential. The difference in the lethal toxicity between Nha 10 (LDs0 = 5.0 #g/g of mouse) and Nha 12 (LDs0= 63/~g/g of mouse) is likely due to the substitution of Gln-7 in Nha 10 to Arg-7 in Nha 12, for all the normal neurotoxins have no charge on residue 7. The substitution of residue 7 in Nha 12 may directly affect the interaction with AChR, because the substitution does not appear to induce a major conformational change in the molecule (Harvey et al., 1984). Trp-29 is also conserved in all neurotoxins sequenced so far. The microenvironment of Trp-29 has been studied by ultraviolet absorption and fluorescence spectroscopy (M6nez et al., 1976, 1980b; Nakanishi et al., 1980). The exchange rate of the N-I hydrogen of the Trp-29 indole ring with the solvent deuterons is reduced only by 10% as compared with free tryptophan, suggesting the indole ring is exposed to the solvent (Nakanishi et al.,

Structure-function relationship of postsynapticneurotoxins

429

1980). N M R studies, show that the N-1 hydrogen of Trp-29 indole ring is close to Asp-31, probably forming a hydrogen bond, in short neurotoxins, Cbt (Endo, T. et al., unpublished results), Nno II (Arseniev et al., 1976), Nmm I (Naja mossambica mossambica) (Lauterwein et al., 1978) and Nmm III (Arseniev et al., 1981a), and long neurotoxins, Tx B and ~-Bgt (Endo, T. et al., unpublished results; see Section 3.2.2.3). In spite of such a conserved interaction between Trp-29 and Asp-31, there has been argument about its role in neurotoxicity in the literature. For some neurotoxins, modification of Trp-29 reduces toxicity (Chang and Hayashi, 1969; Seto et al., 1970; Karlsson et al., 1973; Chang and Yang, 1973: Allen and Tu, 1985), but for other toxins it does not significantly alter the toxicity (Chicheportiche et al., 1972; Karlsson et al., 1972; Ohta and Hayashi, 1974b). Dv 4.7.3 (Dendroaspis viridis), which has the same amino acid sequence as Dv 4.9.3 except for having Trp-29 oxidized, shows ~ toxicity of Dv 4.7.3 (Table 3). Thus, Trp-29 appears to be important in expressing neurotoxicity. Position 33 is invariably occupied by aromatic amino acid residues, Phe, His or Trp. The Br~nsted plots of the H - D exchange of the C-2 protons of His-4 and His-33 in Cbt have revealed that both residues are exposed to the solvent (Endo et al., 1979). The results of photooxidation (Huang et al., 1972) and the N M R photochemically induced dynamic nuclear polarization (photo-CIDNP) study (Muszkat et al., 1984) of Cbt also suggest that His-33 in Cbt is not buried in the molecule. The pKa of His-33 imidazole ring was found to be 5.5-6.0 in Cbt (Endo et al., 1979), Nno II (Arseniev et al., 1976) and Nmm III (Arseniev et al., 1981a). His-33 thus has no charge at neutral pH and therefore His-33 in some neurotoxins provides a hydrophobic group similar to that of Phe-33 or Trp-33 in other neurotoxins. Lc a and Lc b with normal lethal toxicity have Thr-33 with a hydrophobic methyl group instead of His/Phe/Trp-33. The selective photooxidation of His-33 in Cbt diminishes the lethal toxicity and antigenic specificity greatly (Huang et al., 1972). Accordingly, although the functional role of His/Phe/Trp-33 is not clear at present, it may well enhance the affinity for AChR through hydrophobic interactions with the binding sites in AChR. The results of chemical modification and sequence comparison have revealed several functionally important residues of neurotoxins in this way, but at the same time they have supported the idea of the toxin-receptor interaction with multiple attachment points. In this picture, no single functionally essential residue may be found to play a strictly crucial role in neurotoxicity in contrast to the case of enzymes with narrowly defined active sites. Indeed, chemical modification of all the amino groups or of all the arginine residues of ~Cbt still leaves neurotoxic activity in the ~-Cbt derivatives (Martin et al., 1983). Guanidination of all the amino groups in Eb left about 10% of the AChR binding activity of native Eb (Hori and Tamiya, 1976; Ishikawa et al., 1977). The loss of each of the functionally essential residues (groups) appears to reduce the AChR binding activity to some extent but not to diminish the activity completely. 4.3.2. C-terminal Tail in Long Neurotoxins Compared with short neurotoxins, long neurotoxins have an additional C-terminal tail segment as long as 8 residues. In the crystal structure of ~-Cbt, considerable disorder is found around the tail region (Walkinshaw et al., 1980). However, interactions between the C-terminal tail segment and the main part of the molecule have been indicated by N M R studies. Inagaki et al. (1981b) have observed NOE between Tyr-69 in the tail and Thr-13, Leu-41 and Phe-43 in the main part of Ls III, and have suggested that the tail part probably stabilizes the triple-stranded E-sheet structure supporting the hydrophobic core of the central loop. The pH dependence of the chemical shift and intensity of Trp-72 N-I proton resonance of As b indicates that Trp-72 in the tail is not fully exposed to the solvent and the Trp-71 N-1 proton resonance is affected by the ionization of probably Glu-39 carboxyl group in the main part of the molecule (Endo et al., 1986b). To elucidate the role of C-terminal tail segment in long neurotoxins, the C-terminal 4 to 5 residues of ~t-Bgt and Lcb were cleaved off by carboxypeptidase P (Endo, T. et al., 1987b). The J.P.T. 3 4 / ~ F

430

T. ENDO and N. TAMIYA

effect of such deletion on the toxin conformation was monitored in the proton NMR spectra and CD spectra. The results indicate that the removal of the C-terminal residues primarily affects the residues close to the cleavage site and does not induce a major conformational change. Therefore, the C-terminal tail of long neurotoxins does not appear to be important in maintaining the specific polypeptide chain folding. On the other hand, competition binding with 3H-labeled Tx ~ to AChR has revealed that the cleavage of the C-terminal residues significantly reduces the binding ability of ~-Bgt or Lc b to AChR. Since the C-terminal tail always contains Lys or Arg, it is likely that the basic residues in the tail segment in long neurotoxins are directly involved in the binding to AChR. In contrast to the results described above, the derivatives of ~-Cbt (Karlsson et al., 1972) or Tx B (Ohta et al., 1976), whose C-terminal 4 residues are removed by carboxy peptidase, still show a lethal toxicity 50-70% that of native toxins. This may be related to the suggestion by Ishikawa et al. (1977) that Tx B binds to AChR in a somewhat different manner from 0t-Bgt. Short neurotoxins have several invariant residues that are not found in long neurotoxins; such residues include Asn-5, Ser/Thr-9, Thr/Ser-39 and Arg-43 (Table 3). Some of these residues in short neurotoxins may well provide alternative receptor binding sites, and these binding sites may enhance the affinity of short neurotoxins for AChR. A new type of neurotoxin, x-Bgt, that binds to neuronal AChR but does not block neuromuscular AChR has been isolated from the venom of Bungarus rnulticinctus (Quik and Lamarca, 1982; Chiappinelli, 1983; see review by Chiappinelli, 1986). ~-Bgt produces different effects from 0t-Bgt, and the neuronal nicotinic AChR has been suggested to be structurally distinct from that of the muscle AChR. x-Bgt consists of 66 amino acid residues and contains 5 disulfide bridges at the same positions as long neurotoxins (Grant and Chiappinelli, 1985). Its amino acid sequence (Table 2) displays high sequence homology with other neurotoxins, especially with long neurotoxins, suggesting a close similarity in the polypeptide chain folding. There are, however, striking differences in the sequence between x-Bgt and other long neurotoxins: x-Bgt does not have invariant or type-invariant residues, Tyr-25, Trp-29, Ala-46 or Val/Ala-52, or an extra C-terminal tail. In particular, the replacement of Trp-29 with Gln-29 and/or the shortened C-terminal segment in xBgt may well switch its biological function, changing the affinity with neuronal AChR and muscle AChR; at least, the lack of (2-terminal tail should reduce the ability to bind to muscle AChR. x-Bgt has also been found to exist as a dimer under the conditions used for electrophysiological experiments, indicating that the dimer may be physiologically active (Chiappinelli and Lee, 1985). 4.4. BINDING REGION OF NEUROTOXINS FOR THE RECEPTOR In the crystalline state of Eb and ~t-Cbt, the side chains of the functionally important residues described above are organized to form a well-defined region on the toxin molecules. Lys-27, Trp-29, Asp-31, Phe/His/Trp-33, (Gly-34), Arg-37 and Lys-53 are located in the three-stranded fl-sheet structure, and their side chains (except for Gly-34) are oriented in the same direction from the concave surface of the disk-like molecule. Such a spatial arrangement of the functionally important residues allows us to speculate on the location of a putative binding region that may interact with the binding sites of the receptor to other agonists and antagonists. As already described, the binding sites for neurotoxins and for agonists or antagonists probably overlap with each other in AChR, but they may not be identical. Besides, at least for a weak interaction with AChR, a strict three-dimensional structure of neurotoxins does not appear to be essential. For example, even if the five disulfide bridges of 0t-Cbt are reduced and alkylated under non-denaturing and mild conditions, the resulting derivative of 0t-Cbt still exhibits weak but definite receptor-binding activity of 0.02% of the native toxin (Martin et al., 1983). A synthetic peptide corresponding to the segment 16-56 of a short neurotoxin from Naja naja philippinensis, Nnp, has a 10-fold higher affinity for AChR than acetylcholine (Juillerat et al., 1982).

d-Tubocursrlne

~CH~

.~.~o.

'

N~

7" c.,

/N~ /

~H~ ~.~c.. ,..-.o ~

CH~

O~N

Tyr-25

--~0 Asp-31

@

/ ~-~ Glu-42 CH~--

~F ~C%

~ NH%~N~c~' ~H~Arg-37 Phe-33

Trp-29

Neurotoxln

~O ~ NH~Arg-37 Asp-31 Phe-33

-~ [" ~

/CH~--NH~

L~-Sa

~

~NH O-~NH~

~CH~

~-~

Trp-

NH, Arg-37

Neurotoxin

//~C--OO Asp-31

/

~

"~

"~...,......,.%....,..

..........

:~ .~

~

.........j

~

~.........

(c)

1 ....

~CC~P~C~SC

T

P~

Y T - I WC D I F *~ M P ~ P T

~s

~T ~ T ~

~

~L~£RK~

A~

~

~

I S

NeurotoxJn (o-Cbt)

E CC NP ACGR HYS C GK

+

GIA

G N S A R K G RSN

=00

+ ....

GRCCHPACGKNYSC E C C N P A C GR H Y S C

0 ....

gII

HI GI

ConotoxJn " Residue

Rabies virus glycop~oteJn

(~)

C-alkaloid E Neurotoxin FiG. 9. (a) Proposed mimic structures o~ a ncurotoxm ~o~ ~ccty]cho]inc (Tscrnogiou ~t GL, 1978), d-tubocurafinc (T~m~ya ~t ~L, 1980), and C-~]ka]oi~ E (Du~ton ~nd Hider, 1980). (b) Amino ~cid ~qucn~s o~ conoto~ns. (c) Common amino ~cid ~qucncc [or r~bics virus g]ycoprotcin ~nd ~ long ncurotoxin (=-C5t). *D~su]fidc brid~cs arc ~o~cd ~twccn Cys-2 and Cys-7 ~nd ~ t w ~ n Cys-3 and Cys-13. t]nwrJant or typc-invafi~nt residues ~or long ncurotoxin~ ~rc underlined.

~

II ~ V ~ ill

~o~c~

"~~

C

~~.C

N/

CH~

~

Acetylchollne

-C--O-- CH~--CH~--N~--CH3 CH,

O

~.

432

T. ENDOand N. TAMIYA

It is, however, still tempting to consider that neurotoxins contain a structure that mimics that of various agonists or antagonists for binding to the receptor. Beers and Reich (1970) have examined space-filling models of agonists and antagonists for both nicotinic and muscarinic AChR. They have proposed that the specific binding of nicotinic agents to AChR is mediated by an electrostatic interaction and a hydrogen bond involving a positive charge and a hydrogen-bond acceptor separated by about 0.59 nm in most of the agents. Acetylcholine has a positive charge at the quaternary ammonium and a hydrogen bond acceptor at the carbonyl oxygen. Tsernoglou et al. (1978) and Low (1979) proposed that Asp-31 and Arg-37, which are on opposite strands of the fl-sheet to each other, may provide a structural mimic of acetylcholine (Fig. 9). They put forward a hypothesis that Arg-37 binds to the choline cation binding site and Asp-31 provides a hydrogen bond acceptor that is an alternative to the carbonyl oxygen of acetylcholine. Upon binding to the receptor, the possible formation of a hydrogen bond between the Asp-31 carboxylate group and the Arg-37 guanidino group would make the geometry of these groups much closer to that of the acetylcholine structure. However, such a hydrogen-bonded ion pair between Asp-31 and Arg-37 is not detected in the free toxin in the crystalline state. N M R analyses of neurotoxins in solution give no indication of the hydrogen bond either, because the pKa (2.5-3.0) of Asp-31 does not appear to be perturbed significantly by a positive charge (Arseniev et al., 1976, 1981a; Bystrov et al., 1978; Lauterwein et al., 1978; Endo, T. et al., unpublished results). Another possibility is the inclusion of Gly-34 instead of Asp-31 for the mimic structure, i.e. the peptide carbonyl group of invariant Gly-34 may play the role of the carboxyl group of Asp-31 (Tsernoglou et al., 1978). It has been noted that the spatial arrangements of several central-loop residues in a neurotoxin resemble those of particular groups and atoms in d-tubocurarine, a potent antagonist for AChR isolated from Chondodendron tomentosum, and C-alkaloid E from Strychnos toxifera (Fig. 9) (Dufton and Hider, 1977, 1980; Tamiya et al., 1980). Carboxylate oxygens of Asp-31 and Glu-42 in a neurotoxin may correspond to triplets of an oxygen atom of d-tubocurarine, positive charges of Lys-27 and Arg-37 to tubocurarine's ternary and quaternary ammoniums, hydrophobic bulky side chains of (Tyr-25,) Trp-29, Phe/His/Trp-33 and Ile-40 to the aromatic rings, respectively. A possible argument against this idea is that, although the geometrical patterns of these elements of a neurotoxin and d-tubocurarine are related to each other, they may not be superimposed precisely in the two molecules in crystals: the distance between Phe-33 and Tyr-25 in Eb and ~-Cbt is much longer than that between the corresponding aromatic rings of d-tubocurarine (Sobell et al., 1972). However, d-tubocurarine has some flexibility in its structure (Reynolds et al., 1975), and a neurotoxin molecule also contains conformational flexibility particularly around the central loop. Molecular dynamic simulations of Eb have suggested that there exist conformational fluctuations that allow the central loop to bend toward the concave side of the molecule (Nakamura, H., personal communication). Accordingly, both d-tubocurarine and the central loop of a neurotoxin are possibly capable of undergoing a conformational change that alters the longitudinal length of the loop to fit with the common binding sites in the receptor. M6nez et al. (1982) have added Lys-53 to the binding region of a neurotoxin to the receptor noting the charge distribution of antagonist tris-onium compounds such as gallamine. Conotoxins MI, GI, GIA, and GII isolated from the venom of the marine snail Conus geographus are peptide neurotoxins that bind specifically to AChR (Gray et al., 1981; Olivera et al., 1985). They consist of 13-15 amino acid residues with an amidated Cterminus and are cross-linked with two disulfide bridges (Fig. 9) (Gray et al., 1981, 1983; Nishiuchi and Sakakibara, 1982). In spite of their smaller molecular sizes compared with those of neurotoxins, they are at least 10-fold more toxic than d-tubocurarine. Thus, the relationship between the three-dimensional structure and function of conotoxins is of great interest because they may carry at least a part of the active site of snake neurotoxins. On the basis of the CD analyses, Hider (1985) has proposed a model for the conformation of conotoxins, which includes an ~-helix in segment 6-11. The author has suggested that

Structure-function relationship of postsynapticneurotoxins

433

the positive charges at the N-terminus and Arg/Lys-9, which are separated by 0.140.15 nm, together with the two aromatics, His-10 and Tyr-1 l, may construct similar structural arrangements to those of neurotoxins (Arg-37, Lys-53 and aromatic residues in positions 29 and 33) and in alkaloid antagonists. Gray et al. 0985) have proposed on the basis of model-building a possible binding region of conotoxin that involves His-10, Arg/Lys-9 and Glu-1 and the ~t-amino group as alternatives to Phe/His/Trp-33, Arg-37 and Lys-53 of a neurotoxin. However, recent N M R studies (Kobayashi et al., 1986) have shown that conotoxin GI in dimethylsulfoxide solution does not take the conformation proposed by Hider 0985). Inter-proton NOEs were measured by two-dimensional N M R spectra and were used as proton-proton distance constraints for the calculation of polypeptide conformations. The conformation obtained indicates that the side chain of Arg-9 is in proximity to that of Asn-4, and that Arg-9 and Asn-4 side chains are on the opposite side to the N-terminus and Tyr-ll side chain of the rather compact molecule. Thus, the binding region of conotoxin for AChR is not deafly identified at present. Neurotropic rabies virus is an enveloped negative-strand RNA virus and the glycoprotein comprising the surface spikes of the envelope attaches to the cell surface by binding to normal cellular constituents, which act as viral receptors. Comparison of the sequence data has shown that there is a significant similarity between the entire long neurotoxin sequence and a certain segment of glycoprotein from neurotropic rabies virus (Lentz et al., 1984). The greatest similarity occurs with the amino acids comprising the segment 25-44 of long neurotoxins, which probably contains receptor binding site (Fig. 9). This finding could be evidence that a host-cell receptor for the rabies virus might be the acetylcholine receptor, and is consistent with the observation that the rabies virus is localized in regions containing a high density of AChR on mouse diaphragms and cultured chick myotubes (Lentz et al., 1982). The structural pattern of the arrangement of functionally important residues may also be analyzed in terms of electrostatic potentials on the molecular surface, which may be important at the first stage of the process of intermolecular recognition between a neurotoxin and AChR. The electrostatic potential on the molecular surface can be visualized using a combination of computer programs for a raster graphic display (Weiner et al., 1982; Nakamura et al., 1984). Figure l0 shows the calculated electrostatic potential on the molecular surfaces (the concave active side toward the viewers) of a short neurotoxin, Eb, and a long neurotoxin, ~-Cbt (Nakamura, H. and Endo, T., unpublished results). The color contour level close to purple indicates the negatively charged region and that close to red the positively charged region on the molecular surface. As can be clearly seen, the potential surface patterns of the putative binding region, the protruding second (center) and third (right) loops are common to these two molecules. The potentials are strongly positive and a pattern of positive, neutral and positive potential regions that align across the tip of the two loops is created by the invariant residues, Arg-37, Asp-31 and Lys-53. In particular, the negative charge of the carboxylate group of Asp-31 surrounded by clustered positive charges in the central loop may serve as a "marker" of the binding region in the neurotoxin molecule that is to be initially recognized by the receptor. Then rigorous conformational adjustment will follow to form a tighter and more stable complex. In contrast, the potential surfaces of the rest of the molecules, namely the parts of the surface of the first (left) loop, the core part and the back, show little resemblance between the two toxins, suggesting that these regions are not primarily responsible for the initial toxinreceptor recognition process common to short and long neurotoxins. 4.5. CONFORMATIONALCHANGEIN THE NEUROTOX~NAND RECEPTOR It is natural to consider that the function of the receptor, such as the opening and closing of the ion channel and desensitization, is accompanied by a conformational transition in the receptor. The conformational change, which is related to the binding of cholinergic agonists, is probably a global one involving more than one subunit, because

434

T. ENDO and N. TAMIYA

the ion channel is thought to comprise the amphipathic part of each of the five subunits (Finer-Moore and Stroud, 1984; Mishina et al., 1985; Young et al., 1985; Criado et al., 1986), and its entrance is rather large compared with the size of cholinergic ligands. Positive cooperativity observed on the binding of cholinergic agonists to AChR (Weber and Changeux, 1974; Neubig and Cohen, 1979; Sine and Taylor, 1980; Fels et al., 1982) can be also ascribed to some allosteric conformational change in several subunits which mediates a cooperative interaction between the two agonist binding sites, because the two 0t-subunits are not adjacent. A line of experimental evidence has accumulated that AChR undergoes a conformational transition upon binding of cholinergic agonists. Conformational transition has been monitored by a fluorescence change of intrinsic tryptophan residues (Gr/inhagen and Changeux, 1976; Bonner et al., 1976; Barrantes, 1978; Kaneda et al., 1982) or by fluorescence probes introduced to the receptor (Griinhagen and Changeux, 1976; Grfinhagen et al., 1977; Quast et al., 1978; Schimerlik et al., 1979; Dunn et al., 1980). Stopped-flow fluorescence measurements have allowed observation of the rapid kinetics of a conformational change. In many cases the rate constants characterizing the conformational transition are too small to account directly for the activation of the ion channel and, therefore, the slow transitions are probably related to the desensitization process of the receptor. However, in some cases, a conformational transition was observed with rate constants high enough to be expected for a rate-limiting step in the process of channel activation(DunnandRaftery, 1982a,b).Aconformationalequilibriumamongdifferentconformational states of AChR, probably corresponding to different functional states, has been characterized by either stopped-flow experiments observing the fluorescence change upon binding of the fluorescent cholinergic agonist Dns-C6-Cho to the receptor (Heidmann and Changeux, 1979, 1980) or quench-flow experiments observing the agonist-triggered ion flux from the Li+-loaded AChR-rich vesicles (Neubig and Cohen, 1980; Heidmann et al., 1983). The binding of small antagonists also induces a conformational transition in the receptor as monitored by fluorescence change, but in these cases different effects have often been pointed out when compared with agonists. For example, the fluorescence change of quinacrine, with a fluorescent probe introduced to the receptor, was observed under conditions of direct illumination upon binding of both agonists and antagonists, but a change in quinacrine fluorescence by energy~ transfer from the receptor was observed only with agonists (Grfinhagen and Changeux, 1976). Neurotoxins are potent antagonists but they differ in the molecular size from small antagonists such as d-tubocurarine. Quinacrine fluorescence did not change on neurotoxin binding, either by direct illumination or by energy transfer (Griinhagen and Changeux, 1976). Some other conformational change in the receptor was, however, suggested upon binding of a neurotoxin. We have already mentioned that a neurotoxin has some flexibility, for example around the putative active binding region, and its local conformation is readily perturbed by changes in the surrounding environment. Such conformational flexibility in a neurotoxin molecule will enhance the conformational matching between the toxin and AChR, while the flexibility of the neurotoxin molecule will increase the entropy of the free toxin molecule. Then, as a compromise, a relatively rigid structure with some flexibility, as found in neurotoxins, will be suitable for efficient protein-protein interactions (Inagaki et al., 1981c). Of course, the situation is also true for AChR, if the receptor molecule has some flexibility. Then such conformational changes of both neurotoxin and receptor molecules will enhance the affinity of the two molecules by an optimum rearrangement of their local structures, and/or allow them to produce a mutually locked complex. Indeed, a conformational change in the receptor upon neurotoxin binding has been suggested by thermodynamic analyses (Maelicke et al., 1977), and by directly monitoring the intrinsic tryptophanyl fluorescence change in the receptor (Endo et al., 1986a). Likewise, a conformational change has been found for a neurotoxin by the observation that the spin-spin interaction in the ESR spectra changes upon binding of the dispin-labeled 0t-Cbt (Bystrov et al., 1983) or the dispin-labeled ~-Bgt (Rousselet et al., 1982) to AChR.

FIG. 10. Stereo view of the electrostatic potentials on the molecular surfaces of Eb (A) and ctCbt (B). The electrostatic potential values are shown with color codes (from purple to red) corresponding to I0 contour levels from - 10 kcal/mole to 40 kcal/mole. The concave active side of the molecule is toward the viewers. The potential surfaces were calculated and displayed by the programs AVEMS and GRAIP, which were developed by H. Nakamura (the University of Tokyo), using the coordinates obtained from the Protein Data Bank (Kimball et al., 1979; Walkinshaw et al., 1980).

435

Structure-function relationship of postsynapticneurotoxins

437

4.6. ESR AND FLUORESCENCEANALYSESTO MONITOR NEUROTOXIN-RECEPTORINTERACTION 4.6.1. Spin-Labeled Neurotoxin Direct identification of the residues of a neurotoxin involved in receptor binding may be made by introducing an ESR spin label or a fluorescence label at the residues concerned and subsequently monitoring their behavior in the binding process. Monospin-labeled derivatives have been prepared for short neurotoxins, Nno II (Tsetlin et al., 1979b) and Tx ~t (Rousselet et al., 1984), and a long neurotoxin, ~-Cbt (Tsetlin et al., 1982), and their ESR spectra were recorded with and without AChR. Spin label was incorporated at the N-terminus, Lys-15, Lys-26, Lys-27, Lys-51, Lys-53, Glu-2 and His-33 of Nno II (Tsetlin et al., 1979b). In the presence of AChR, ESR signals of each derivative broadened to varying extents reflecting the change of mobility of spin label after receptor binding. The data were treated by the isotropic rotation model to compare the mobility by the use of correlation time in the free state and in the bound states. The results show that spin labels at the N-terminus, Glu-2, Lys-26, Lys-27 and His-33 decrease their mobility significantly, probably due to the contact with the receptor surface. The solvent accessibility of spin labels was estimated by examining the effect on ESR signals by adding paramagnetic ions. Although the results are not independent of the effect of the paramagnetic probes used, Fe(CN)~-or Nt(Ac)2, " 2 + they indicate that the paramagnetic-ion accessibility of the spin labels is considerably diminished for Lys-27 and Lys-53 in the toxin-receptor complex as compared with the free toxin. Accordingly, Lys-27 and Lys-53 are shielded from the paramagnetic ion in the toxin-receptor complex, but the mobility of spin label at Lys-53 is not restricted in the complex. It is interesting to note that the side chains of Lys-27, His33, Lys-53 are accommodated in the putative binding region in the neurotoxin, but that the N-terminus and the side chains of Glu-2 and Lys-26 are out of this region. Employing a similar approach, the effect of receptor binding was monitored in the ESR spectra for five Tx ~ derivatives, which were monospin-labeled at the N-terminus, Lys-15, Lys-27, Lys-53 or Lys-57 (Rousselet et al., 1984). In the absence of receptor-rich membrane, spin label incorporated at the N-terminus, residues 15, 53 and 57 have their own rapid motion, while that at Lys-27 has no residual mobility but reflects overall tumbling of the toxin molecule. In contrast to the case of Nno II, the motion of the spin label bound to Lys-53 was greatly reduced in the presence of receptor-rich membrane, and at the same time, accessibility to paramagnetic Ni ~+ ion was also decreased for this spin label. These discrepancies could be due to the introduction of the rather bulky group of a spin-label as well as the modification of a charged group of neurotoxins may perturb the binding properties of neurotoxins to AChR. Interactions of a mono- and dispin-labeled long neurotoxin, ~t-Cbt, with acetylcholine receptor were monitored by ESR spectroscopy (Tsetlin et al., 1982). ~-Cbt was monospinlabeled at Lys-27, or dispin-labeled at Lys-27 and Lys-15, Lys-27 and Lys-39, Lys-27 and Lys-53, Lys-27 and Lys-73, or Cys-30 and Cys-34. The spin label at Lys-27 reduced its accessibility to paramagnetic ions on receptor binding, and a less pronounced shielding effect was observed for Lys-53, which was greatly shielded from paramagnetic ions in the case of short neurotoxins.

4.6.2. Fluorescent-Labeled Neurotoxin Tsetlin et al. (1979b, 1982) introduced a dansyl group in a short neurotoxin, Nno II at the ~-amino group of the N-terminus or at the e-amino groups of Lys-26, Lys-27 or Lys53. The binding of Lys-53-dansylated toxin to AChR was accompanied by a change in fluorescence spectrum (excited at 330 nm), a blue shift of the emission maximum and an increase in fluorescence intensity, indicating a transfer of the label into a more hydrophobic environment. The increase in fluorescence intensity was larger when excited at 297 nm

438

T. E~DOand N. TAM1YA

(absorption band of tryptophan residues) and conversely, intrinsic tryptophanyl fluorescence of AChR was quenched on binding of Lys-53odansylated toxin when excited at 330 nm. Therefore, energy transfer occurs between the dansyl label of the toxin and tryptophan residues of AChR in the complex. Similar indications of a transfer of the dansyl label into a hydrophob.ic environment were observed on toxin-receptor binding for Lys27-dansylated Nno II and more weakly for Lys-26-dansylated Nno II, while no change in the fluorescence spectra was observed for the' dansylolabeled derivatives at the N-terminus and Lys-I 5 of Nno II. Thus, in the toxin-receptor complex, Lys-27 and Lys-53 are likely to be in close contact with the receptor surface, and also Lys-26 on the opposite side of the molecule is near the receptor molecule. These results are consistent with those obtained in the parallel ESR studies using spin-labeled Nno II derivatives described above (Tsetlin et al., 1979b). Johnson and Yguerabide (1985) carefully estimated the contribution of the geometrical masking of the fluorophore and the translational and rotational mobilities of the labeled toxin, to the fluorescence quenching on binding of N,-fluorescein isothiocyanate-Lys-27 ~Cbt to AChR (Cheung et al., 1984). They have reached a different conclusion from that of Tsetlin et al. (1979b, 1982), that is, Lys-27 is exposed to the bulk solvent both when the labeled toxin is free in solution and when it is bound to AChR. Johnson et al. (1984) also measured the fluorescence energy transfer between the two types of fluorescently labeled ~-Cbt, N,-fluorescein isothiocyanate Lys-27 ~-Cbt and monolabeled tetramethylrhodamine isothiocyanate ~t-Cbt, bound to AChR. Larger energy transfer was found to occur between two toxins on separate receptor molecules than between two toxins on two sites on the same receptor molecule. The calculated distance between the two fluorophores indicates that they lie on the outer perimeter of the receptor rather than near the central axis of the receptor.

4.6.3. Stopped-Flow Fluorescence Measurement Kaneda et al. (1982) have observed that the intrinsic fluorescence of AChR purified from N a r k e japonica is decreased upon binding of agonists, while antagonists like dtubocurarine do not affect the fluorescence intensity. AChR from Narke japonica also exhibits a significant fluorescence change upon neurotoxin binding (Endo et al., 1986a). This fluorescence change arises primarily from a conformational change of AChR, and reflects the binding process of the toxin to AChR. By using the stopped-flow technique, the time dependence of the fluorescence has been monitored for various neurotoxins (Endo et al., 1986a). The binding process of a neurotoxin and AChR involving a conformational change in AChR may be described by either of the two following schemes. A conformational change may occur either prior to or subsequent to the toxin binding: Mechanism I kl

AChR. k

AChR + N t x .

" AChR 1

k2

" AChR. Ntx

(3)

k- 2

Mechanism II AChR+Ntx,

kI k- I

•AChR.Ntx,

k2

~AChR.Ntx

(4)

k_ 2

We consider the conformational change in AChR only to avoid complexity, and AChR represents the receptor after a conformational transition. In mechanism I, AChR in the absence of a neurotoxin is in rapid equilibrium between two conformational states, and a

Structure-functionrelationshipof postsynapticneurotoxins

439

neurotoxin binds only to the state AChR. In mechanism II, the fast binding of a neurotoxin to the receptor induces a slow conformational transition in the receptor. Both mechanisms I and II show monophasic kinetics for the binding of a neurotoxin and AChR, if the preequilibrium isomerization in mechanism I or the bimolecular binding in mechanism II is sufficiently rapid. Of course, we cannot exclude the mechanism (instead of mechanism I) that a free neurotoxin is in a rapid conformational equilibrium and AChR binds only to a small fraction of the conformational states of a neurotoxin. However, this seems unlikely because the residues of a neurotoxin that control the apparent rate of the fluorescence change are on the surface of the neurotoxin molecule (Endo et al., 1986a) and they appear hardly to be involved in the conformational equilibrium, if present, of a neurotoxin molecule itself. Stopped-flow fluorescence measurements upon toxin-receptor binding have revealed that rate constants for fluorescence changes are larger for short neurotoxins than for long neurotoxins, although the distribution ranges of the rate constants for short and long neurotoxins overlap each other (Endo et al., 1986a). The tendency of short neurotoxins to associate with AChR much faster than long neurotoxins was also pointed out by Chicheportiche et al. (1975). The faster association and dissociation rates of short neurotoxins with the receptor led to the suggestion by Hayashi et al. (1981) that short neurotoxins are more useful than long neurotoxins as the adsorbent for affinity chromatography in the isolation procedure of AChR. Previously, on the basis of the rather limited data available, we proposed a hypothesis that the overall structural rigidity, reflected in the amide proton exchange rates, of long and short neurotoxins is related to the reversibility of the receptor binding, the more rigid toxins being the less reversible (Endo et al., 1981). However, no correlation was found for the variation of rate constants and structural rigidity for the toxins within a group of short neurotoxins (Endo et al., 1986a). Bystrov and co-workers claimed that the enhanced flexibility in long neurotoxins might be related to the less reversible binding of long neurotoxins to the receptor (Bystrov et al., 1983). However, as already described (see Section 3.3), there is no obvious difference in the conformational flexibility between short and long neurotoxins. Instead of these hypotheses, it is more likely that the differences in the local structure and/or binding sites for AChR (see Section 4.3.2) between short and long neurotoxins may be responsible for the different rates of their association with AChR. Indeed, differences in the toxin-receptor interactions of long and short neurotoxins were inferred from the different distribution ranges of the steady-state fluorescence change (Endo et al., 1987a). The relationship between the rate constants of a fluorescence change of the short neurotoxins and their amino-acid sequences, thermal stability, hydrogen-deuterium exchange behavior as indexes for structural rigidity, overall net charge and so on has been examined (Endo et al., 1986a). A rule was found that if a toxin lacked a positive charge at' one of the two residues 27 and 30, the rate of fluorescence change was reduced greatly. It is interesting to note that residues 27 and 30 are expected to lie in the anti-parallel fl-sheet structure and their side chains are oriented in opposite directions. The side chain of residue 27 is on the proposed binding surface of the toxin molecule, while that of residue 30 is on the opposite surface. Therefore, in the transition or final state of the receptor binding process, the neurotoxin molecule probably embeds, at least, the protruding mainchain loop into the narrow cavity of the receptor molecule, allowing both sides of the flsheet structure to be buried. Further, it has also been found that the overall net charge of short neurotoxins affects the toxin-receptor binding process (Endo et al., 1986a); the more positive a toxin is, the faster it associates with the receptor and decreases the fluorescence. In other words, the net charge of the neurotoxin molecule probably plays an important role in the process of interaction with the binding sites in AChR which are expected to have several negative charges. J.P.T. M/~--F*

440

T. ENDO and N. TAMIYA

4.7. SPECIES DIFFERENCE IN THE RECEPTOR AND ITS INTERACTION WITH NEUROTOXIN

The recent cloning and sequence analyses of fish electric organ AChR (Noda et al., 1982, 1983a,b; Claudio et al., 1983; Sumikawa et al., 1982; Devillers-Thiery et al., 1983), mammalian AChR (Noda et al., 1983c; Tanabe et al., 1984; Takai et al., 1984, 1985; Kubo et al., 1985; Shibahara et al., 1985; LaPolla et al., 1984) and avian AChR (Nef et al., 1984) have shown a high degree of amino-acid sequence homology among various species. Such a high sequence homology has allowed the production of functional hybrid AChR molecules composed of subunits from, for example, Torpedo AChR and rat or calf muscle AChR in Xenopus oocytes (White et al., 1985: Sakmann et al., 1985). However, species differences in the sensitivity of neuromuscular preparations to neurotoxins have been reported (Burden et al., 1975; Lee and Chen, 1976). It is known that animals are sometimes resistant to the neurotoxin they produce, and this appears true for snakes also. Burden et al. (1975) have observed different effects of a long neurotoxin, ~-Bgt, and a short neurotoxin, Cbt, on skeletal neuromuscular junctions of various animals, including frogs, lizards and snakes. ~-Bgt was found to be more effective than Cbt, probably due to the more irreversible binding of ~-Bgt to AChR. The synapses of snakes (Henophidea, and Caenophidea) were less sensitive than those of lizards and frogs. Interestingly, synapses both from frog and ribbon snake (Caenophidea) were blocked by tubocurarine. Takasaki and Tamiya (unpublished results) have examined the effects of injection of Eb to various animals, from fish to mammals. The injection of Eb killed Redsword tail (,Yiphophorus hellerri), Japanese frog ( Rana nigromaculata), tortoise ( Seudemys scripta), chick and mouse effectively (LDs0 values are 0.1~).3 #g/g of body weight). On the other hand, 440- to 1500-fold amounts of Eb as compared with the LDs0 value for mice failed to kill various kinds of snakes; LDs0 values were estimated to be larger than 0.22mg/g for Japanese Mamushi, Agkistrodon blomhoffi blomhoffi, 0.066mg/g for Himehabu, Trimeresurus okinavensis, O. 17 mg/g for Japanese grass snake (Yamakagashi), Rhabdophis tigrinus tigrinus, and 0.23 mg/g for Japanese racer (Shimahebi), Elaphe quadrivirgata. In these cases, tubocurarine was also found to act as effectively on snakes as on other animals. The difference in the sensitivity of neuromuscular junctions to neurotoxins or in the LDs0 values for Eb are probably due to the differences in the structure of AChR molecules in the animals themselves. AChR of snakes may sterically prevent the access of neurotoxin molecules to the binding sites or they may lack certain residues that are a part of the neurotoxin binding sites, while the binding sites may be still open to small agonists and antagonists. Then it would be of interest to find the size dependence, if present, of the effect of antagonists on their receptor binding, by testing the effect of the peptidic neurotoxins, conotoxins, whose molecular sizes are between those of tubocurarine and snake neurotoxins. Lee and Chen (1976) have tested the reversibility of the neuromuscular block produced by different neurotoxins in the muscle of various species including rat, kitten, chick and frog. The neuromuscular blocks caused by short neurotoxins, Cbt or Eb, were generally reversible when the toxin was removed by repetitive washing, while a long neurotoxin, ~Bgt, gave less reversible effects. This can be explained by the slow rates of ~-Bgt for the association and dissociation reactions with AChR (Ishikawa et al., 1977, Endo et al., 1986a). A species difference was found in the susceptibility and reversibility of the neuromuscular block for the different nerve-muscle preparations, but the results are rather complicated. The susceptibility of the frog muscle to neurotoxins differs appreciably from one species to another; Cbt and Eb produce reversible neuromuscular blockiug in the muscles of the kitten, rat and frogs (Rana tigrina and Rana narina) (Lee and Chen, 1976), slowly reversible blocking in the chick (Harvey and Rodger, 1978), but irreversible in the frog, Rana plancyi (Lee and Chen, 1976).

Structure-function relationship of postsynaptic neurotoxins

441

5. CONCLUDING REMARKS

From the starting point of the crystal structures of three neurotoxins, research on neurotoxin structure has developed into the elucidation of conformational properties of various neurotoxins in solution. Conformations of the neurotoxins have been compared in solution and in crystal, and it has been shown that the crystal structures alone are not sufficient to describe the function of a neurotoxin completely. In some cases, the conformation of a neurotoxin in solution under physiological conditions is not identical with that found in the crystal; in other cases, flexibility is necessary for the neurotoxin molecule to undergo a conformational change in the process of toxin-receptor interaction. In spite of the detailed knowledge available on the structures of neurotoxins themselves, much of the molecular mechanism of neurotoxins still remains unclear. Each step of the toxin-receptor interaction--i.e, the binding of neurotoxin to AChR, conformational changes in neurotoxin and AChR, and the resulting non-depolarizing block of neuromuscular transmission--poses many questions, which are also linked to the function of AChR itself. The investigation to resolve such problems should provide deeper insight into the molecular explanation of neurotransmission in the nerve-muscle systems and also should contribute to the understanding of protein (or peptide)-receptor interactions in general. Acknowledgements--The authors express their gratitude to Prof. T. Miyazawa (the University of Tokyo) for fruitful discussions, to Prof. K. Hayashi (Gifu Pharmaceutical University), Prof. F. J. Joubert (National Chemical Research Laboratory), Prof. M. Z. Atassi (Baylor College of Medicine), Prof. V. F. Bystrov (M.M. Shemyakin Institute of Bioorganic Chemistry), and Prof B. W. Low (Columbia University) for providing information contributing to this article, to Prof. D. Mebs (University of Frankfurt) for sending us the compilation of the amino acid sequences of neurotoxins and other snake venom proteins. List of Biologically Active Components from Snake Venoms, to Dr. Y. Mitsui (the University of Tokyo) for providing the program for the molecular graphics STDRAW and that for solvent accessibility ACCESS, to Dr. H. Nakamura (the University of Tokyo) for providing unpublished information on molecular dynamics simulations and making pictures of Fig. 10 by the computer programs AVEMS and GRAIP, and to G. Kawai (the University of Tokyo) for transferring the graphics data. The authors are grateful to Dr. R. C. Hider (University of Essex) and Dr. A. M6nez (CEN, Saclay) for providing Fig. 4 prior to the publication. T.E. would like to thank Prof. M. Oya (Gunma University) for his discussion and encouragement.

REFERENCES A~;A~I~, D. A. and S'n~ot~, R. M. (1982) ~t-Bungarotoxin structure revealed by a rapid method for averaging electron density of non-crystallographically translationally related molecules. Acta Cryst. A38: 186-194. ALL,S, M. and Tu, A. T. (1985) The effect of tryptophan modification on the structure and function of a sea snake neurotoxin. Mol. Pharmacol. 27: 79-85. ,aatsv.mt,v, A. S., B^LASHOVA,T. A., U~ra~, Yu. N., TS~TLI~,V. I., BYS~OV, V. F., IWNOV, V. T. and Ovcm~m~ov, Yt~. A. (1976) Proton-nuclear-magnetic-resonance study of the conformation of neurotoxin II from Middle-Asian cobra (Naja naja oxiana) venom. Eur. J. Biochem. 71: 595-606. A~s~m~v, A. S., PAsm~ov, V. S., PLgZI-~,atCOV,K. A., ROCnAT,H. and BYSTROV,V. F. (1981a) The tH nuclearmagnetic-resonance spectra and spatial structure of the Naja mossambica mossambica neurotoxin III. Eur. J. Biochem. 118: 453-462. A~s~m~v, A. S., UTKIS, Yt~. N., PASm~OV,V. S., TSETLI~q,V. I., l w s o v , V. T., BvS~OV, V. F. and O v c m ~ l ~ o v , Yv. A. (1981b) 19F NMR determination of intramolecular distances in spin- and fluorine-labelled proteins: neurotoxin II Naja naja oxiana. FEBS Lett. 136: 269-274. B~LLIWT, M., P~'mCK, J., Lm, J. and HE~eMA~qt4,S. (1982) Molecular cloning of cDNA coding for the T-subunit of Torpedo acetylcholine receptor. Proc. Natn. Acad. Sci. U.S.A. 79: 4466-4470. B^sr~s, B. E. C, MILr~, R. and Sm~OLn~l,R. A. (1974) The primary sequences and neuromuscular effects of three neurotoxic polypeptides from the venom of Dendroaspis viridis. Eur. J. Biochem. 45: 457-468. B~s~, E. A., WmocOWSl~l, J. and Cnro, T. H. (1971) Cholinergic receptor molecules and cholinesterase molecules at mouse skeletal muscle junctions. Nature (London) 234: 207-209. BAm~,~'r~s, F. J. (1978) Agonist-mediated changes of the acetylcholine receptor in its membrane environment. J. Mol. Biol. 124: 1-26. B~cms, G., G ~ s ~ , C., v^r4 Rx~rscHoa~s, J., Joy,R, E., ROCnAT,H. and Mw.A~r),~,F. (1976) Purification of six neurotoxins from the venom of Dendroaspis viridis: primary structure of two long toxins. Eur. J. Biochem. 68: 445-456. BEEr,S, W. H. and R~CH, E. (1970) Structure and activity of acetylcholine. Nature (London) 228: 917-922. BLANCHARD,S. G., QUAST,U., REED,K., LEE,T., SCm~,m~L~K,M. I., VANDLEN,R., CLAUD10,T., STRADER,C. D,, MOO.~E, H.-P. H. and RAF~Y, M. (1979) Interaction of ~I-~-bungarotoxin with acetylcholine receptor from Torpedo californica. Biochemistry 18: 1875-1883. BONN~, R., BA~AWrm, F. J. and .Iovx~, T. M. (1976) Kinetics of agonist-induced intrinsic fluorescence changes in membrane-bound acetylcholine receptor. Nature (London) 263: 429-431.

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