Prediction of tertiary structures in ‘scorpion-toxin’ type proteins

Life Sciences, Vol. 50, pp. 683-693 Printed in the USA

Pergamon Press

PREDICTION OF TERTIARY STRUCTURES IN "SCORPION-TOXIN' TYPE PROTEINS P. Narayanan and K. Lala Department of Life Sciences, University of Bombay, Vidyanagari, Bombay 400 098, India. (Received in final form January 6, 1992)

Summary In the three-dimensional architecture of macromolecules, the structural stability and proper folding manifest due to cooperative packing interactions of various segments. Hydrophobicity is the major facto!~ stabilizingprotein-protein associations. In the disulfide-containing proteins, S-S bonds are integral part of structural motifs and large part of the protein-folding problem can be reduced to identifying and understanding motifs and subdomains of these proteins. Identifying such a motif with S-S bonds in "scorpion-toxin' type proteins, and from model-building studies, five tertiary structural models for these type of proteins can be proposed. These canonical structural models can be refined by regular minimum energy and computer simulation methods to arrive at the final tertiary structures. Such "models' can be of considerable use i) in understanding the biochemical reaction mechanisms in the structure-function relationships, ii) structure determination by X-ray methods (molecular replacement method), iii) drug design etc. The cherished goal of molecular biophysicists, structural biologists and others, is to predict the tertiary structures (three-dimensional folding) of macromolecules from their primary structures. Prediction of secondary structural elements, namely a-helix and I~-sheets, from the amino acid sequence data can be attempted by various methods, and prevalent method being that of Chou-Fasman (1). Prediction of final tertiary structure from the sequence data of protein alone is extremely difficult and met with little success even though there are many structure prediction-methods available (2). Information on protein folding cannot be deciphered from the aminoacid sequences alone, because folding is a highly cooperative process. One of the ways of simplifying this complex problem is to classify proteins by shapes and motifs and then carry out model building of proteins based on these shapes by employing energy minimization methods to obtain final "predicted' tertiary structures with no steric strain. This means, in majority 0024-3205/92 $5.00 + .00 Copyright o 1992 Pergamon Press plc All rights reserved.

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of cases, the protein folding problem can be reduced to identifying and characterizing the motifs and subdomains. This method is one or t w o steps below in the hierarchy of predicting final structure, but the logic has sound structural basis due to the fact that three-dimensional structures of proteins (folding/packing) are conserved in evolution more than protein primary structures (3). Even in the cases where no obvious sequence homologies may be found, the unknown structures can be considered from the structural motifs and subdomains. Aggregates of secondary structures is called motif. One example is the coiled-coil in which t w o e-helixes wind around each other to form a supercoil. Other examples of structural motifs are ~ , I~el~, Rossmann-fold, I~-meanderetc. The empirical rules that govern the packing that occurs between and among secondary structures ( a-helixes and I~-sheets), to form motifs and the three-dimensional (tertiary) structures are:

1) Residues that become buried in the interior of a protein close-pack. That is, hydrophobicity is the major factor stabilizing protein-protein associations. 2) Associated secondary structures retain conformation close to the minimum free energy conformation of the isolated secondary structures. That is, the conformational angles of the main chain and the side chains of the polypeptides lie in the narrowly allowed regions of Ramachandran map (4). These principles imply that the secondary structures found in proteins interact in a manner that gives the maximum van der Waals energy and induces no appreciable steric strain (5). One way of circumventing, in the "structure-prediction' methods, the problem of cooperative process in folding is to incorporate the knowledge (empirical rules) of folding patterns in disulfide-containing proteins. Such empirical rules are of much help in the case of disulfide-containing proteins because S-S bond peptide pair contains secondary and tertiary structures similar to native protein. The protein-folding problem, as a kinetic problem, has been addressed (6,7) by analyzing the intermediates of disulfide-containing proteins. Best example of such studies is that of bovine pancreatic trypsin inhibitor (BPTI). It is a protein with 58 amino acids and three S-S bonds (C5 - C55; C30 - C51 & C14 - C38). It has ordered segments; t w o e-helixes (2-7 & 4 7 - 5 5 ) and t w o I~-sheets (14-25 28-37 & 43-46). Structural patterns of various intermediates established that,

1) C30-C51 bond (single S-S intermediate occurs in all f o l d i n g patterns

(C30-C51 connects the middle of I~-strand 28-37 & a-helix 47-55; that is, sheet-helix stabilization).

2) C30-C51 & C5-C55 intermediates (two S-S bond intermediate) is the

most stable and has the properties of the native s t r u c t u r e (C5-C55 connects helix 1 and helix 2; that is, helix-helix stabilization). The crucial role of subdomains in the folding of BPTI has been pointed out (8), Methods

In all the disulfide-containing proteins, S-S bonds are found as integral parts of structural motifs and are not just involved in structural stability but also in creating hydrophobic moieties-an entropy factor. The same underlying principles of structure stability, by S-S bonds are discernible from the threedimensional structure of scorpion toxin, CsE V3, CsE V2, CsE V (9,10) and

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685

AaH II (11) by X-ray diffraction methods. The most prominent structural features consmst of a dense core of secondary structural elements in which an u-helix (residues 23-32) is cross-linked through two disulphide bridges (C25-C46 and C29-C48) to the middle strand of a three-stranded anti-parallel S-sheet moiety (residues 1-4; 37-41 and 46-50; that is helix-sheet stabilization). The other two S-S bonds are involved in stabilizing strand-loop and loop-loop interactions. NMR studies on variant-3 (CsE V3) in solution are in agreement with the crystal structure data (12). In all these structures the structural motif of C25-C46; C29-C48 moiety is retained; shown schematically in Fig. 1 and Table I (Residue numbering corresponds to CsE V3).

25

29 X-- X-- X - - ~ - - - ~ 262728/ ~/~

!x../

<

FIG. 1 Structural motif of C25-C46; C29C48 moiety (CsE V3 numbering is retained throughout).

46 47 48

TABLE I Secondary structuralelements atthe C25-C46 and C 2 9 - C 4 8 m o i e t y

(A)

X26-X27-X28

I)

"CsEv3" type has DTE; NEE; DGL; NAD; NDL; GRE; DKL etc.

zz)

a-helix. "AaH-IT" type has NNQ

(i.e.

(a-helix region)

small hydrophobic,

(can s u s t a i n

a-helix),

neutral

or h y d r o p h i l i c

can sustain

s a m e as I case.

III) "Bom I" type has DTL (can s u s t a i n a-helix),

s a m e as I case.

Iv)

"I5A" type has -RD, -RA, -AD, -AT

v)

"CnII-II" type has SKP, LPK, XP(Proline, helix b r e a k e r p r e s e n t ; c a n n o t s u s t a i n a-helix); Apamin (Bee venom toxin) and MCD (Mast cell d e g r a n u l a t i n g peptide) ARm and RKI (can s u s t a i n a-helix).

(B)

X'

(can s u s t a i n

a-helix),

(47) R e s i d u e

I) "CSEV3" type II) "AaH-IT" type III)'"Bom I" type IV) "IsA" type V) "CnII-11" type

s a m e as I case.

(B-strand region)

X' = W or Y X' = Y X'= u n k n o w n X'= L X' = K

(Hydrophobic residues) (Hydrophobic) s a m e as I (Partially s e q u e n c e d ) (Hydrophobic) s a m e as in I (Hydrophilic) d i f f e r e n t f r o m I

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Tertiary Structures in Toxins

vol. 50, No. I0, 1992

Number of S-S bonds vary from four or three in scorpion venom toxins to t w o in bee venom toxin apamin and mast degranulating (MCD) peptide. Based on the knowledge discerned from the empirical rules governing the packing of secondary structural elements and structural features from the three-dimensional structures of scorpion venom toxins, structural hierarchy of the disulfide bonds in these "scorpion toxin' type proteins, whose primary structures are available, can be established with t w o core' disulfide bonds, with strong neighborhood correlation, stabilizing the structural motif (Fig.1 and Table I). Further, the basic underlying logic comprisesi) in disulfide-containing proteins, the S-S bonds render not only structural stability but also functional features as well, ii) secondary structures and motifs play the central role in protein folding and iii) packing interactions stabilize protein folding and, therefore, tend to be conserved in protein folds. Therefore, identifying and characterizing the hierarchial features of motifs and subdomains in these proteins will go a long way in unravelling the protein-folding problem and predicting th~ -final' structures. Structural data by NMR studies on Buthus eupeus Mq (13), honey-bee venom apamin (10) and scorpion insectotoxin I~A (14-16) ahd representation of polypeptide chain folds (polypeptide backbone) of CsE V3, Mq and I~A (1 7) as well as other "scorpion-toxin' type proteins lend credende to tl~is logic- that is, to the retention of this structural motif with t w o S-S bonds in all the "scorpion-toxin' type proteins (Table II). From the available structural data by X-ray diffraction and NMR methods, it is apparent that the three-dimensional structures of the scorpion insectotoxins (9,10), I~A (15,16) and neurotoxin MQ (13) show the same conformational motif in their spatial organization, that i~, similar orientation of the a-helix relative to the I~sheet. Particularly close correspondence is seen between the spatial structures of neurotoxin M , and insectotoxin CsE V3, although they share a poor sequence homology (3 3). This experimental evidence reinforces the statement made earlierin the text, "Even in the cases where no obvious sequence homologies may be found, the unknown sequence can be considered from structural motifs and subdomains". This has been borne out from the wealth of structural data on various proteins and has become an axiom in structureprediction methods. In relation to this structural motif (Fig. 1) other S-S bonds can be predicted. From such a prediction the tertiary structures of all scorpion venom toxins, apamin and MCD peptide (1 8) that have been sequenced to date, can be classified uf~,der five "canonical' structural categories (Figs. 2-6 and Table II). Listed in Table III are amino acid sequences of some of the toxins from Table II, to highlight the distribution of secondary structure elements (ahelix and I~-sheets) representative of these categories. CsE V3 is retained as a standard in all categories and residue numbering is according to this protein. As highlighted in the Table III, the secondary structural elements are in general found in the same regions in all categories as found in category I. Apamin and MCD peptide have t w o disulphide bonds forming the structural motif that is found in other scorpion toxins; however, the sense of direction is different (10). All the same, the ~xistence of these structural elements need not be exactly in register (compare the position of a-helix region of 15A, which belongs to category IV with that of the toxins belonging to category I and the content of secondary structural elements can vary for different categories. Nor can it be emphasized that the existence of these secondary structural elements is conditional for their spatial organizations, though in ~eRneral the condition is valid. As pointed out in Table I, Apamin which has R in the a-helix region (C-X-X-X-C) can sustain a-helix and indeed has an a-helix (residues 9-16), whereas sore3 of the toxins that belong to the same category (category V) contain proline in this region, which is a helix breaker

m

FIG. 2

-- 5

1.'

-'

°1"

FIG. 4

FIG. 6

Born I t y p e s t r u c t u r e

""-"

~'~

Cn I1-11 t y p e s t r u c t u r e

%

structure

FIG. 3 A a H IT t y p e

5,

FIG. 5

•

".e'.__d

15A t y p e s t r u c t u r e

CsE V3 t y p e s t r u c t u r e

"",.

1

'

•

00

m

I-'-

o

11 (D W

(3

CA ¢'t I1

I=

I't ¢1"

O

O

O

O

688

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TABLE II Structural categories in scorpion toxins II)"CSE V3 Type" AaH I AaH I' AaH I'' AaH AaH Amm Amm Amm AaH I0 Ii 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 3O 31 32 33 34 35 36 37 38 39 4O 41 42 43 44 45 46

II III III V VI IT4

Be I2 B e M9 Be MI0 Bom IV Bom VI Bop I B o p II Bot I B o t II B o t III Bot VIII B o t XI Bt -II Ce II-6.3 Cll II.6 CII II.9 Clt 1 Cn II-9.2.2 Cn II-13 Cn II-14 CsE I CsE Vl C s E V2 C s E V3 CsE V Css I C s s II C s s III L q q III Lqq IV Lqq V Ts F Ts III-8 Ts IV-5 Ts IV-6 Ts II-9B Ts II-10

Reference I 23 24 24 11,25 26 24 27 24 21 28 13 29 30 30 24 24 30 30 30 24 24 31 32 33 33 34 35 35 35 36 37 10,37 10,38 i0 39 39 40 24 41 42 43 43 43 40 40 4O

II)"AaH-IT Type" 1 2 3

Reference I

AaH-IT AaH-ITI AaH-IT2

44 45 22,45

III)"Bom I Type"

Reference

1

1

Bom

I

IIV)"I5A Type" 1 2 3 4

I5A A m m P2 B e Ii Bs

I

b

30

Reference I 15,16 24 46 47

IV)"Cn II-ll Type" Reference I Cn II-ll Cn II-10.2 Ts II-9A Apamin MCD (Mast cell degranulating peptide)

48 48 48 10,49 18

AaH;Androctonus australis Hector, Amm;A.m.mauretanicus, Be;Buthus eupeus, Bom;B.o.mardochei, Bop;B.o.tunetanus, Bot; B.o.paris, Bs; B.sindicus, Bt; B. tamulus, Ce; Cen truroides elegans, CII; C. I. limpidus, CIt; C. I. tecomonas, Cn;C.noxius, CsE;C.sculpturatus Ewing, Css;C.s.suffusus, Lqq;Leiurus q. quinquestriatus, Ts; Tityus serrulatus.

CsE V3 CsE Vl CsE V2 CsE I CsE V AaH I A a H I' A a H I'' A a H II AaH III A a H IT4 Amm V Be M 9 B e M I0 Be I 2 Bom IV Bot I B o t II Bot XI Cn II-14 C s s II Css III Lqq IV Lqq V Ts T Ts If-10 Bt-II

I)Cs_E V 3

Toxin

(S-S

bonds: 12

or

N-terminal

C25-C46; 16

sequence

sequence

C29-C48; C16-C41 & C12-C65) 25 29 41 46 48 65 B a 6 6 --KEGYLVKKSDGOKYGCLKLGENEGCDTECKAKNQGGSYGYOYAF ..... ACWC-EGLPESTP-TYPLPNKS-C --KEGYLVKKSDDCKYDCFWLGKNEHCNTECKAKNQGGSYGYCYAF ..... ACWC-EGLPESTP-TYPLPNK--CS --KEGYLVNK-TGCKYGCLKLGENEGCDKECKAKNQGGSYGYCYAF ..... ACWC-EGLPESTP-TYPLPNK--CSS --KDGYLVNK-TGCKKTCYKLGENDFCNRECKWKHIGGSYGYCYGF ..... GCYC-EGLPDSTQ-TWPLPNK--CT -KKDGYPVDSG-NCKYECLK---DDYCNDLCLER--KADKGYCYWG-K--VSCYCY-GLPDNSP-TKT-SGK--CNPA -KRDGYIVYPN-NCVYHCVPP ..... CDGLCKKN-GGSSGSSC-FLVPSGLACWC-KDLPDNVP-IKDTSRK--CT -KRDGYIVYPN-NCVYHCIPP ..... CDGLCKKN-GGSSGSSC-FLVPSGLACWC-KDLPDNVP-IKDTSRK--CT -KRDGYIVYPN-NCVYHCVPP ..... CDGLCKKN-GGSSGSSC-FLVPSGLACWC-KDLPDNVP-IKDTSRK--CTR -VKDGYIVDDV-NCTYFCGR---NAYCNEECTKL-KGESG-YCQWASPYGNACYCYK-LPDHVR-TKG-PGR--CH -VRDGYIVNSK-NCVYHCVPP ..... CDGLCKKN-GAKSGS-CGFLIPSGLACWC-VALPDNVP-IKDPSYK--CHS --EHGYLLNKYTGCKVWCVIN--NEECGYLCNKRRGGYYG-YCYFW-KLA--CYC-QGARKSEL-WNYKTNK--CDL -LKDGYIIDDL-NCTFFCGR---NAYCDDECKKK-GGESG-YCQWASPYGNACWCYK-LPDRVS-IKE-KGR--CN -ARDAYIA-KPHDCVYECYNPKG-SYCNDLCTENGAE--SGYCQILGKYGNACWCXQ-LPDNVP-IR-IPGK--CN -VRDAYIADDK-DCAYFCGR---NAYCDEECKK--GAESG-KCWYAGQYGNACWCYK-LPDWVPIKQKVSGK--CN --ADGY-VKGKSGCKISCFL--DNDLCNADCKYY-GGKLNSWCIPD-KSG-YCWCPNKGWNS---IKSETNT--CN -GRDAYIAQPE-NCVYECAK---NSYCNDLCTKX-GATSG-YCXW ....... C.C ................... C -GRDAYIAQPE-NCVYECAQ---NSYCNDLCTKQ-GATSG-YCDWLGKYGNACWC-KDLPDNVP-IRI-PGK--CHF -GRDAYIAQPE-NCVYECAK---NSYCNDLCTKN-GAKSG-YCQWLGRWGNACYC-XDLPDKVP-IRI-EGK--CHF -LKDGYIVDDR-NCTYFCGT---NAYCNEECVKL-KGESG-YCQWVSRYGNACWCYK-LPDHVR-TVQ-AGR--CG --KDGYLVDAK-GCKKNCYKLGKNDYCNRECRMKHRGGSYGYCYGF ..... GCYCEG-LSDSTP-TWPLTNKT-C --KEGYLVSKSTGCKYECLKLGDNDYCLRECKQQYGKSSGGYCYAF ..... ACWC-THLYEQAV-VWPLPNKT-CN --KEGYLVSKSTGCKYECLKLGDNDYCLRECKQQYGKSSGGYCYAF ..... ACWC-EALPDHTQ-VWV-PNKT-CT GVRDAYIADDK-NCVYTCGS---NSYCNTECTKD-GAESG-YCQWLGKYGNACWCXK-LPDKVP-IRI-PGK,-CR -LKDGYIVDDK-NCTFFCGR---NAYCNDECKKK-CGESG-YCQWASPYGNACWCYK-LPDRVS-IKE-KGR--CN --KEGYLMDH-EGOKLSCF-IRPSGYCGRECGIKKG-SSG-YOAW--P---ACYCY-GLPNWVKVWDRATNK--C --KEGYLMDHE-GCKLSCF-IRPSGYCGRECGIKKG-SSG-YCAW--P---ACYCY .................. C --EDGYLLNRDTGCTVSCGT ...... C-RYCND ......... C ......... C.C ................... C

TYPE

Complete

Amino acid sequence comparison of "scorpion-toxin' type proteins.

TABLE III

38 37 37 36 l0 23 24 24 25 26 21 27 13 29 28 30* 30 30 24 35 39 40 41 42 43 40* 31"

Reference

m

0 X

0 m O--

M

0

O

O

o

Bom

C25-C46; 25 65 B ..... ACWC-EGLPESTP-TYPLPNKS-C ..... CYCFGLNDDKKVLEISDTRKSYCDTTIIN ..... CYCFGLNDDKKVLEISDTRKSYCDTTIIN ..... CYCFGLNDDKKVLEISDTRKSYCDTPIIN

& C42-C65) 46 48

TYPE

(S-S b o n d s :

C25-C46;

C25-C46;

49 18

38 48 48* 48*

38 15,16 24 46 47

38 30*

38 44 45 45

* : partially sequenced, # : structural motif similar to other scorpion toxins but sense of direction is different (Numbering for Apamin is also indicated) AaH;Androctonus australis Hector, Amm;Androctonus m.mauretanicus, Be;Buthus eupeus, Bom;Buthus o.mardochei, Bs;Buthus sindicus, Bt;Buthus tamulus, CsE;Centruroides sculpturatus Ewing, Cn;C.noxius, Css; C.s.suffusus, Lqq;Leiurus q. quinquestriatus, Ts; Tityus serrulatus.

II-ll

(S-S b o n d s :

C29-C48; & C16-C41) ~ 5 5 C s E V3 --KEGYLVKKSDGCKYGCLKLGENEGCDTECKAKNQGGSYGYCYAFACWCEGLPESTPTYPLPNKSC Cn II-ll TIINVKC---TSPKQCSKPCKELYGSSAGAKCMNGKCKCBN Cn II-10.2 TFIDVKC---GSSKECXP ......... Ts II-9A VFINAKC--RGS-PECLPKCKEAXGKAAGKCXN ...... 5 ~ 15 1234 Apamin # APETALCARRCQQH -CNCK MCD-Peptide # RHVIKPHICRKICGKN KCNCK

V)Cn

~5 A TYPE

I T Y P E (S-S b o n d s : C 2 5 - C 4 6 ; C 2 9 - C 4 8 ; C 1 6 - C 4 1 & C 4 - C 1 2 ) B ~ B B --KEGYLVKKSDGCKYGCLKLGENEGCDTECKAKNQGGSYGYCYAF ..... ACWC-EGLPESTPTYPLPNK---SC -GRDCYIAQPE-NCVYHCF-PGSHG-CDTLCKEK-GATSG-XCGFLPGXXVAC.C .................... C

(S-S b o n d s : 12 16

C29-C48; C16-C41 & C12-C30) ~ B B C s E V3 KEGYLVKKSDGCKYGCLK--LGENEGCDTECKAKNQGGSYGYCYAFACWCEGLPESTPTYPLPNKSC I5A MCMP-CFTTDPNMAKKC-RDCC .... GG-NGKCFGPQCLCNR A m m P2 -CGP-CFTTDPYTESKC-ATCC .... GG-RGKCVGPQCLCNRI Be I 1 MCMP-CFTTRPDMAQQC-RACC .... KG-RGKCFGPQCLCGYD Bs peptide I RCKP-CFTTDPQMSKKC-ADCC---GGG-KGKCYGPQCLC

IV)

C s E V3 Bom I

III)

TYPE

C29-C48; C16-C41 29 41 B a ~ --KEGYLVKKSDGCKYGCLKLGENEGCDTECKAKNQGGSYGYCYAF -KKNGYAVDSS-GKAPECL-L--S~VZCNNQCTKV-HYADKGYCCLLS -KKNGYAVDSS-GKAPECL-L--SB~ZC~ECTKV-HYADKGYCCLLS -KKDGYAVDSS-GKAPECL-L--SNYCYNECTKV-HYADKGYCCLLS

AaH-IT

C s E V3 A a H IT AaH IT1 A a H IT2

If)

o

o M m m

m

m

= o

m

o

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Tertiary Structures in Toxins

691

(then the mhelix should be off-register as in the case of 15A). As has already been stressed, it is not just to the sequence homology, nor to the existence of secondary structural elements, that more weightage should be given but to the structural motifs, structural modules and subdomains in the structure prediction procedures.

Results and Discussion Applying the empirical rules governing the packing interactions between and among various secondary structural segments and w i t h the incorporation of the hierarchy of S-S bonds in stabilizin 9 the structural motifs, the structure prediction methods, of predicting tertiary structures from their sequences, can be made more reliable in the case of disulfide-containing proteins. From such a study structures of all the "scorpion-toxin' type proteins, can be classified under five tertiary structure categories (canonical structures) (19,20). Available amino acid sequence and structural data lend support to the necessity and use of such a classification t h a t has been proposed. For example the scorpion venom toxin AaH IT4 from Androctonus australis Hector was suggested to be a member of a new class of toxins (21). However, from our predictive methods of structural characterization of "scorpion-toxin' type proteins, it should be classified under the structural category I, the most common type, and does not necessitate invokin 9 any new class for this toxin. The secondary structure analyses by circular dlchroism (CD) of this toxin indicate same amount of secondary structural elements (e-helix 13% ; I~-sheet 23% that are found in CsE V3 and AsH II (14% and 22% respectively) whose three-dimensional structures have been determined by X-ray diffractions methods. These values are similar in other structures also belonging to the category I.In contrast, the NMR data on AaH IT1 and CD data on AaH IT2, which should fall in the category II (AaH IT type) according to our classification, showed marked difference in their structure content (e-helix 20% and I~-sheet 30%) in comparison to the structures of the category I (21). Minimum energy methods, computer graphics and simulation can now be employed to refine these "predicted' canonical structural models to obtain final tertiary structures. Work is underway in this direction. There are no X-ray structural data available on toxins of other categories except for preliminary X-ray crystallographic data on AaH IT2 (22) (which belongs to category II of our classification). Though available spectroscopic data on other categories support our categorization, high-resolution structural information by two-dimensional NMR and X-ray diffraction methods would greatly help in ascertaining the correctness of this hypothesis and the "predicted structures. Conversely, the "predicted structures can be used as "hypothetical' models in the structure determination by 2D-NMR and by X-ray diffraction method (by molecular replacement procedure). Such attempts are underway to solve the structure of AaH IT2 (category II) by "molecular replacement' method using "hypothetical' model based on the three-dimensional structures of the CsE V3 (10) and AaH II molecules (11) which belong to category I. Acknowledgements The financial assistance from the Department of Science and Technology, Government of India, grant (SP/SO/D37/88) is acknowledged. The help rendered by the Distributed Information Center (DIC), Indian Institute of Science, Bangalore, India is appreciated.

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