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Antibiotic Peptides
C H A P T E R
Colicins: Bacterial/Antibiotic Peptides O. SHARMA, S. D. ZAKHAROV, AND W. A. CRAMER
tor specific for that type of colicin. Indeed, as was confirmed later, colicins enter cells by parasitizing receptors in the outer membrane whose physiological purpose is the binding and import of metabolites (e.g., vitamins, sugars, metals such as iron). Subsequently, several colicins differing in their mode of action (discussed in [34]) and the receptors they parasitize to gain entry into target cells, have been identified. Ribbon diagrams of those colicins (la, N, B, which are pore formers and E3, an rRNase), whose 3-D structure has been solved, are shown in Fig. 1. Colicins have been classified according to the translocation system that they appropriate to enter the bacteria. Group A colicins utilize the bacterial Tol-dependent translocation system consisting of the proteins TolA, TolB, TolQ, TolR, and Pal (Table 1). The name of these proteins is derived from the fact that their mutants are "tolerant" to colicin action. Group B colicins utilize the Ton-dependent system, consisting of TonB and ExbB-ExbD (Table 2). "Ton" mutants are resistant to the phage Tl—hence the name "Ton." As can be seen from the tables, the lethal action of most colicins is exerted either by cellular de-energization [22, 26] that results from the formation of a highly conducting pore [29] in the cytoplasmic membrane [12], or by an enzymatic nuclease digestion mechanism [1, 23, 24].
ABSTRACT The functions of receptor binding, cytotoxicity, and translocation across the cell envelope, necessary for colicins to be imported into the cell and to exert their lethal effect on the cell, are encoded in defined peptide domains of the colicin polypeptide. The major lethal effects of the colicins are exerted through pore formation and intracellular nuclease activity. High-resolution x-ray structures of four colicins and x-ray/NMR structures of four C-terminal domains encoding cytotoxic function are known. Cells producing colicin are protected against autocytotoxicity by a -lOkDa immunity protein, which is a tightly bound soluble component of the nuclease colicins or a membrane-bound peptide present in low abundance in cells producing poreforming colicins. Import into the cell is guided by a transenvelope network of "tol" or "ton" proteins. DISCOVERY AND CLASSIFICATION [28] Colicins are plasmid-encoded bacteriocins, produced hy Escherichia co/z under stress conditions, which are cytotoxic to closely related strains that contain the required outer membrane receptor(s) but do not produce the cognate immunity protein. Colicins were first discovered in 1925 by Gratia who showed that one strain oi E. coli ("V" for virulent) released a substance found in the filtrate that was toxic to a sensitive strain. Gratia also showed that colicin-resistant mutants could be derived from sensitive strains. Fredericq later suggested that resistance to colicins involved the loss of a surface recepHandbook of Biologically Active Peptides
STRUCTURE OF THE PRECURSOR mRNA/GENE Colicin-producing cells are protected from its cytotoxic action by coordinate synthesis of an immunity 115
Copyright © 2006 Elsevier
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TABLE 1. Colicins that are imported via the Tol system. Although these colicins have a common theme of import into target cells, the receptors and translocators utilized by these colicins are different. They also differ in their cytotoxic activities. Colicin A^ E1 N E2 E7 E9 E3 E6
Activity Protein^
Immunity Protein^
Receptor
Translocation
Cytotoxic Activity
592 522 387 581 566 582 551 551
178 113 131 86 87 86 85 85
BtuB BtuB OmpF BtuB BtuB BtuB BtuB BtuB
OmpF, TolQRAB TolC, TolQRAB TolQRA OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB
Pore forming Pore forming Pore forming DNase DNase DNase 16SrRNase 16SrRNase
^Length In amino acids. ^Sequence of Colicin A from Citrobacter freundii. Tol-dependent colicins E4, E5, E8, K, U, 28b, and DF13 have not been included in the table. Modified from [20].
TABLE 2. Colicins that are imported via the Ton system. Although the mechanism of import of these colicins into the target cells are similar, the cytotoxic activity differs. A common feature of these colicins is the presence of a TonB box that interacts with the TonB protein which transduces energy from the inner membrane to these import processes [27]. Activity Protein^
Immunity Protein^
Receptor protein
B D
510 697
175 87
FepA FepA
la lb
626 626
111 115
CIr Cir
Colicin
Activity Channel Protein synthesis^ Channel Channel
^Length in amino acids. ^Inhibits protein synthesis by cleavage of arginine tRNAs. Ton-dependent colicins Js, M, 5 and 10 have not been included in the table. Modified from [6].
protein that acts specifically against that colicin. Immunity proteins are small (MW -lOkDa) polypeptides that protect the colicin-producing cells from both endogenous and exogenous colicins [5,15]. In the case of pore forming colicins, a small number of immunity molecules (-10^-10^) in the cytoplasmic membrane are able to provide immunity against a functional and lethal channel in that membrane [45]. Regions of homology between the immunity proteins of pore-forming colicins A, B and N have suggested that extramembrane loops LI and L3 of the 3-4 TM helix protein interact with the respective colicins. Thus, mutations in these regions affect the immunity activity of these proteins [11]. A lysis protein localized in the periplasmic space functions to release colicin from producing cells to the extracellular medium, both for colicin-immunity
protein complex of enzymatically active colicins and for pore-forming colicins. In pore-forming colicins, the Imm protein remains in the cytoplasmic membrane [3]. The gene encoding colicin, its cognate immunity protein, and the lysis protein form an operon that is regulated by the SOS (DNA damage-induced) promoter [8]. In the absence of induction, the immunity gene is expressed by a constitutive promoter that provides basal levels of immunity protein for protection against exogenous colicins. THE DOMAIN CONCEPT There are three defined steps in the killing of a sensitive E. coli by most colicins, each of which is carried
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES
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A: H2N
COOH
FIGURE 1. (A) Generic domain structure of colicin polypeptides (top): T, translocation; R, receptor binding; C, catalytic or activity containing domain (B-E). Ribbon diagrams of structures of pore-forming colicins, la (B), B (E), and N (D), and of rRNase colicin E3 (C). Note the prominent long (210 A and 100 A in colicins la and E3) coiled-coil structure of the R-domain. Structures, predominantly a-helical, have also been obtained of the C-domains of (i) colicins A and El by x-ray diffraction, and (ii) of E7 and E9 by solution NMR. The structures of E3 and E7 contain bound immunity protein, as is characteristic of the nuclease colicins. (See color plate.)
out by a separate domain of the colicin that occupies approximately one-third of the polypeptide (Fig. lA; [24]). The first step involves the central "R" (receptor) domain, which mediates the binding of colicin to its outer membrane receptor. The N-terminal "T" (translocation) domain mediates the second step of translocation from the outer membrane receptor to the colicin target in the cell. Finally, the carboxy-terminal "C" (catalytic) domain functions in cytotoxicity. SEQUENCE COMPARISONS (Fig. 2) Groups A and B have subgroups with extended regions of high homology. In group A, colicins with endonuclease activity (E2-E9) have a high degree of homology in the T and R domains (residues 1-449) based on 85% similarity (70% for E7). The subgroup of pore-forming colicins (El, A, and N) have low homol-
ogy between thvemselves and with colicins E2-9 (-13% between any two pore-forming group A colicins) in the T and R domains. However, the homology is higher between C-domains of pore-forming colicins (-30%), which correlates with a common motif consisting of a 10-helix globular structure including a hydrophobic helical hairpin. The subgroup of endonuclease colicins has high homology in the C-domain between IGsRNase colicins E3 and E6 (90%), and between DNase colicins E2, E 7 a n d E 9 (80%). A conserved pentapeptide sequence "TolB box" is present in most of the Tol-dependent colicins. This TolB box has been shown to interact with the TolB protein [4, 30], defining a step in the translocation process. A site of proteolytic cleavage between TR- and C-domains (R447) has been defined in E7 [32]. In group B, the pore-forming colicins la and lb use the CirA iron transport receptor for recognition [6, 7]. They are identical in the T and R domains including
Group A
E7 : E3 : E6 : E2 : E9 : El: A: N:
1 21 41 61 i l l I MSGGDGRGHNSGAHNTGGNI NGGPTGLGGNGGASDGSGWSSENNPWGGGS GSGVHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGLGVGGGASDGSGWSSENNPWGGGS GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGLGVGGGASDGSGWSSENNPWGGGS GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGLGVGGGASDGSGWSSENNPWGGGS GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGIGVSGGASDGSGWSSENNPWGGGS GSGIHWGGGSG METAVAYYKDGVPYDDKGQVIITLLNGTPDGSGSGGGGGKGGSKSESSAAI HATAKWSTAQL -MPGFNYGGKG DGTGWSSERGSGPEPGGGSHGNSGGHDRGDSSNVGNESVTVMKPGDSYNTPVv^GKV MGSNG ADNAHNNAFGGGKNPGIGNTSGAGSNGSASSNR GNSNGWSWSNK
E7 : E3 : E6 : E2 : E9 : El: A: N:
81 101 121 I I I HGNGGGNSNSGGGS NSSVAAPMAFG- -FPALAAPGAGTLGISVSGEALSAAIADIFAALKGPFKFSAWGIALYGI HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG- -FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGV HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG- -FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA KKTQAEQAARAKAAAEAQAKAKANRDALTQRLKDIVNEALMQAEDE-RLRLAK AEEKARKEAEAAR IINAAGQPTMNGTVMTADNSSMVPYGRGFTRVLNSLVNNPLMVQSG-NLPPGYIAJLSNGKVMTEV REERTSGGGGKNV PHKNDGFHSDG SYHITFHGDNNSKPKPGTITPDN G
E7 : E3 : E6: E2 : E9 : El: A: N:
141 161 181 201 I I I I LPSEIAKDDPNMMSKIVTSLPAETVTNVQVSTLPLDQATVSVTKRV TDWKDTRQHIAWAGVPMSVPWNAKPTR LPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE GGLAVSISAGALSAAIADILPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE GGLAVSISAGALSAAIADILPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE GGLAVS ISASELSAAIAGILPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE HNASRTPSATELAHANNAAEKAFQEAEQRRKEIEREKAETERQLKL AEAEEKRLAALSEEAKAVEIAQKKLSA SPAGQNGGKSPVQTAVENYGNERTWTVKVPREVPQLTASYNEGMRIRQEAADRARAEANARALAEEEARAIASGKSKAEF NSGNRGNNGDGASAKVGEI S
E7: E3: E6: E2: E9: El: A: N:
221 241 261 281 I I I I TPGVFHASFPGVPSLTVSTVKGLPVSTTLPRGITEDKGRTAVPAGFTFGGGSHEAVIRFPKESGQKPVYVSVTDVLTPAQ RPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQ RPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP RPGVFTASIPGAPVLNISVNNSTPEVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP RPGVFTASIPGAPVLNISVNDSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP AQSE V^/KMDGEIKTLNSRLSSSI--HARDAEMKTLAGKRISIELAQAIREEKQKQVTASETRINRINS DAGKRVEAAQAA INTAQLIWNNLSGAVSAAN- -QVITQKQAEMTPLKNELAAAJST^^DTKQNEINAAVANRDALNN KPGR YISSNPEYSLLAKLIDAES P DSNIDKMRVDYVN-
E7 : E3 : E6 : E2 : E9 : El: A: N:
301 321 341 361 I I I I VKQRQDEEKRLQQEWNDAHPVEVAERNYEQARAELNQANKDVARNQERQAKAVQVYNSRKSELDAAN VKQRQDEENRRQQEWDATHPVEAAERNYERARAELNQANEDVARNQERQAKAVQVYNSRKSELDAAN KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN AKYKELDELVKKLSPRANDPLQNRPFFEATRRR-VGAGKADITQIQKAISQVSNNRNAGIARVHEAE QRVQETLKFI NDPIRSRIHFNMRSGL- IRAQHSQLSQAmJILQNARNEKSAADAALSAATAQRLQAEAALRAA - - IKGTEVYT FHTRKGQYVKVTVWKGPKYNNKLVK RFVSQFLLFRKE
E7 E3 E6 E2 E9 El
381 401 I I KTLADAKAEI —KQFERFAREPMAAGHRMWQMAGLKAQRAQTDVNNKKAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE ENLKKAQI^nSTLLNSQIKDAVDATVSFYQTLTEKYGEKYSKMAQELADKSK GKKIGN FIGURE 2, Amino acid residues of colicins E3 and la are numbered in groups A and B, respectively. Residues considered to be similar are: D and E; K and R; N and Q; S and T; I, L, and V. Coloring: red (bold)—similar residues In all or most (except one) of sequences. Blue (bold)—similar residues in poreforming colicins (E1, A, N) of group A. Orange and green—used to show similarity within subgroups of DNase and RNase colicins of group A. Green and blue—used to show similarity within subgroups of CirA- and FepAdependent colicins of group B. Alignment was done using the MUSCLE program.
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES A: N-
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AEAAEKARQRQAEEAERQRQAMEVAEKAKDER- - ELLEKTSELIAGMGDKIGEHLGDKYKAIAKDIADNIKNPQGKTIRS EKEKNEK- -EALLKASELVSGMGDKLGEYLGVKYKJSrVA^^^^ 421 I
E7: E3 : E6 : E2: E9: El: A: N:
441 I
461 I
KSDADVALSSALERRKQKENK-EKDAKAKLDKESKRNKPGKATGKGKPVlSnsrKWLNNAGKDLGSPVPDRIANKL KSDADAALSSAMESRKKKEDK-KRSAENNLNDEKNKPRKG FKDYGHDY-HPAP KTENIKG KSDADAALSSAMESRKKKEDK-KRSAENKLNEEKNKPRKG VKDYGHDY-HPDP KTEDIKG KSDADAALSAAQERRKQKENK-EKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKN KSDADAALSAAQERRKQKENK-EKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKS WTEALAAFEKYKDVLNKKFSK/iDRDAiraALASVKYDDWAKH LDQFAKYLKITGHVSFGYDWSDILKI FDDA^mSLNKITM^IPAI4KIOTCADRDAL^/HAWKHVDAQD^lA^IK LGNLSKAFKVADWMKVEKVREKSIEG YNEAJ^ASLNKVLANPKt^KVNKSDKDAIVNAWKQVNAKDMANK IGNLGKA,FKVADLAIKVEKIREKSIEG 481 I
501 I
521 I
E7 : E3 : E6: E2 : E9: El : A: N:
F — DDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKA PKTRTQDVSGKRTSFELHHEKPISQNGGVYDMDNISWT L--GDLKPGI PKTPKQNGGGKRKRWT GDKGRKIYEWDSQHGELEGYRASDGQHLGSFD L--GELKEGK PKTPKQGGGGKRARWY GDKGRKIYEWDSQHGELEGYRASDGQHLGSFE F--DDFRKKFWEEVSKDPDLSKQFKGSNKTNIQKGKA PFARKKDQVGGRERFELHHDKPISQDGGVYDJyOSINIRVTT F--DDFRKAVWEEVSKDPELSKNLNPSNKSSVSKGYS PFTPKNQQVGGRKVYELHHDKPISQGGEVYDMDNIRVTT KDTGDWKPLF LTLEKKAADAGVSYWALLF5S LLAGTTLGIWGIATVTGILCSYIDKNKLNTIN YETG^JWGPLM LEVESIWLSGIASSVALGIFSATLGAYALSLGVPAIAVGIAGI -LLAA\7VGALIDDKFADALN YNTGNWGPLL LEVESVfl IGGVVAGVAISLFGAVLSFLPIS -GLAVTALGVIGI -MTISYLSSFIDANRVSNIN
E7: E3: E6: E2: E9:
541 I PKRHIDIHRGK PKTGNQLKGPDPKRNIKKYL PKTGNQLKGPDPKRNIKKYL PKRHIDIHRGK PKRHIDIHRGK
El: A: N:
EVLGI NEIIRPAH NIISSVIR
la: lb: B: D:
1 21 41 61 I I I I MSD PVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIWGGNEVvVDDSPTRSDIEKRDK MSD PVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIVvGGNEV\IVDDSPTRSDIEKRDK MSDNEGSVPTEGIDYGDTMWWPSTGR-IPGGDVKPGGSS-GLAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKEL MSDYEGSGPTEGIDYGHSMWWPSTGL-ISGGDVKPGGSS-GIAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKEL 81 I
Group B
101 I
121 I
141 I
la: lb: B: D:
EITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADITRQEFRLLQAELREYGFRTEIAGYDA EITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADITRQEFRLLQAELREYGFRTEIAGYDA EEGDWSGWSVSVHSPWGNEKVSAARTVLE NGLRGGLPEPSRPAAVSF EEGDWSGWSVSVHSPWGNEKVSAARTVLE NGLRGGLPEPSRPAAVSF
la: lb: B: D.
161 181 201 221 I I I I LRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQNADKKAA--DMLAEyERRKGILDTRLSELEKNGGAALAVLD LRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQMADKKAA--DMLAEYERRKGILDTRLSELEKNGGAALAVLD ARLEPASGNEQKI IRLMVTQQLEQVTDIPASQLPAAGNNVPVKY RLTDLMQNGTQYMAIIG ARLEPASGNEQKI IRLMVTQQLEQVTDI PASQLPAAGNNVPVKY RLMDLMQNGTQ YMAIIG
la: lb: B: D:
241 261 281 301 I I I I AQQARLLGQQTRNDRAISEARNKLSSVTESLNTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIWSGDPR AQQARLLGQQTRNDRAISEARNKLSSVTESLKTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIV\;SGDPR GIPMTV PWDAVPVPDR--SRPGTNIKDVYSAPVSPNL--PDLVLSVGQ!4NTPVRSNPEIQ GIPMTV PWDAVPVPDR- - SRPGTNIKDVYSAPVSPNL- -PDLVLSVGQMNTPVLSNPEIQ 321
FIGURE 2. (Continued)
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361
381
119
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CHAPTER 18
la: lb: B: D:
I
I
401 la: lb: B: D:
I
441 I
421
AVNSARNNLS ARTNE QKHANDALNALLKEK ENIRNQLSGINQKIAEEKRKQDELKATKDAI - N F AINSARNIWSARTNE QKHANDALNALLKEK E.NIRSQLADINQKI AEEKRKRDEINMVPCDAI -KL AENNAKDDFRVKKEQ ENDEKTVL . TKTSEVIISVGDKVGEY AENKZOCDDFRVKKEEAVARAEAEKAKAELFSKAGVNQPPVYTQEMMERANSVMNEQGALVLNNTASSVQLAM^^^ 461 I
la: lb: B: D:
481 I
501 I
TTEFLKSVSEKYGAKAEQLARE]yL2iG QAKGKKI RI^TVEEALKTYEKYRADIN TSDFYRTIYDEFGKQASELAKELAS VSQGKQI KSVDDALNAFDKFRNNLN LGDKYKALSREIAENINNF QGKTI RSYDDAMSSINKLMANPS AGDIAGNISKFFSNALEKVTIPEVSPLLMRISLGALWFHSEEAGAGSDIVPGm^LEAMFSLSAQMLAGQGWIEPGATSV 521
la: lb: B: D:
I
FAGTIKITTSAVIDNRANLNYLLSHSGLrJYKRNILNDRNPVVT-EDVEGDKK FAGTIKITTSAVIDNR.2U^JL^^yLLTHSGLDYKRNILNDRNPVVT-EDVEGDKKIYNAE:VAEWDKLRQRLLDARNKITSAE5^ EDGVISET GNYVEAGYTMSS NNHDVIVRFPEGSGVSPLYISAVE ILDS-NSLSQRQE EEGVIAET GNYVEAGYTMSS NNHDVIVRFPEGSDVSPLYISTVE ILDS -NGLSQRQE
541
I
561
KKINAKDRAAIAAALESVKLSDISSNLNRFSRGL6YAGKFTSLADWITEFGKAVRTENWR.P KKYNIQDRMAISKALEAINQVHMAENFKLFSKAFGFTGKVIERYDVAVELQKAVKTDNWRP LKINATDKEAIVNAWKAFNAEDMGNKFAALGKTFKAADYAIKANNIREKSIEGYQTGNWGP NLPVRGQLINSNGQLALDLLKTGNESIPAAVPVLNAVRDTATGLDKITLPAV^/GAPSRTILVNPVPQPSVPT-DTGNHQP 581
I
la: lb: B: D:
LEV KTETII AGN/\ATALVALVFSILTGSA FPV KLESLA AGRAASAVTAWAFSVMLGTP LML EVES V^A/--ISGMASAVALSLFSLTLGSALIA EGLS VPVTPVHTGTEVKSVEMPVTTITPVSDVGGLRDFIYVJRPDAAGTGVEAVYVMLNDPLDSGRFSRKQLDKKYKHAGDFGIS 601
621
la: LGIIGY GLLIvmVTGALIDESLV--EKANKFWG 1 lb: VGILGF AIIMAAVSALVNDKFI--EQVNKLIG 1 B: ATWGF VGWIAGAIGAFIDDKFV--DELNHKII K D: DTKKNRETLTKFRDAIEEHLSDKDTVEKGTYRREKGSKVYFNPNTMNWIIKSNGEFLSGWKINPDADNGRIYLETGEL
FIGURE 2. (Continued)
most of the coiled-coil structure (residues 1-426), and have a 60% similarity in the C-domain. CoHcins B and D containing 510 and 627 residues, respectively, act through pore-forming and DNase activities, and use the ferro-enterobactin receptor FepA for recognition [6]. They have a high degree of homology (95%) in the Nterminal segment (res. 1-310). The homology of the aligned segment of central R- and C-terminal domains, excluding that unique to colicin D, is smaller (-30%). Homology between any three of the four colicins of group B aligned in Fig. 2 is 25%. A common feature of the Ton-dependent colicins is the presence of a "TonB box" pentapeptide sequence in a position proximal to the N-terminus [31]. This sequence is present not only in the Ton-dependent colicins, but also in the outer membrane receptor proteins whose function is dependent on the Ton system.
X-RAY STRUCTURES OF COLICINS (Figs. IB-E) (A) The four complete or incomplete structures of colicins that have been solved by x-ray diffraction of three-dimensional crystals and their major properties are summarized: (i) The structure of residues 23-39 and 83-624 of the 626 residue pore-forming colicin la ("B-type," translocated by the Ton system) has been solved to 3.0 A resolution (Fig. IB; [40]). (ii) The structure encompassing residues 84-551 of the 551 residue "A-type" (translocated by the Tol system) endoribonuclease colicin E3 has been solved to 3.0 A (Fig. IC; [35]). The x-ray structure of a complex of the BtuB receptor with bound coiled-coil R-domain of colicin E3 [18], which is relevant to the mechanisms of cellular import, is discussed below, (iii) 292 residues, 93-385,
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES
of a 321 residue chymotryptic fragment of the 387 residue pore-forming colicin N, has provided a 3.1 A structure that contains the globular channel forming domain (C, in blue, Fig. ID) but is missing a substantial part of the T- (and possibly of the R-) domain [38]. (iv) The structure of most of the 511 residue poreforming colicin B (Ton-dependent) (Fig. IE) was solved at 2.5 A resolution [14]. The x-ray and NMR structures of the C-domains of pore-forming colicins A [25] and El [10] show a bundle of 10 a-helices with a hydrophobic helical hairpin anchor at the core. Structures of the endonuclease domain of colicins E3, E7, E9 (Toldependent), and Ton-dependent colicin D in complex with Imm or nucleic acids have also been solved [13, 17, 36]. The T-, R-, and C-domains in colicins E3 (RNase; Tol-dependent) and la (pore-forming; Ton-dependent) are structurally defined (Figs. IB, C) and connected by long a-helices that form coiled-coils, whereas in colicins B (pore-forming; Ton-dependent; Fig. IE), and probably in N (pore-forming; Tol-dependent; Fig. ID), the T- and R-domain functions are located in a globular domain which is separated from the C-domain by a single long helix, forming the shape of a dumbbell. These two distinct shapes are not related to either the import pathway (Tol- or Ton-dependent), or to the mechanism of cytotoxicity (pore-formation or endonuclease). It has been proposed that the coiled-coil structure in colicin E3 is necessary for delivery of T- and C-domains of a receptor-bound colicin to an outer membrane porin through which the C-domain, at least, is translocated to the periplasm [18]. However, involvement of outer membrane porins in the import of Tondependent colicin la has not yet been implied. (B) T-domains. The N-terminal T-domain of colicin la contains a "TonB box" (E23-V27) that is necessary for translocation across the periplasm as part of an antiparallel helix bundle. This secondary structure of the T domain of colicin la contrasts with the p-sheet "jelly roll" that forms most of the colicin E3 T domain. The N-terminal region of the colicin E3 T domain contains a DGSGW (D35-W39) "TolB box" needed for translocation [4] and is glycine-rich (34 of 79 residues), accounting for the disorder in this region of the structure. A possible reason for the difference in the Tdomain structures of colicins la and E3 is that the immunity protein is tightly bound to the T domain in the latter. The N-terminal domain of colicin B is composed of 17 P-strands, 2 a-helices, and long loops. The central region (residues 130-291) of its sequence shares some similarity with the T domain of colicin E3, but the folding pattern does not fit to a jellyroll motif. In both colicins la and B, the N-terminal segments, containing the TonB box (residues 23-27 and 17-21 in la and B, respectively) are folded back and interact with antipar-
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allel helices of the coiled-coil and T-domain in colicin la, and with the N-terminal lobe (TR-domains) of colicin B. (C) R-domains. The 104 residue region at the tip of the 160 A coiled-coil, consisting of an amphipathic twostranded P-hairpin folded around the tip-terminal helix of the coiled-coil was proposed to bind to the colicin la Cir receptor [40]. The R-domain of colicin N was proposed to consist of a 65 residue (residues 97-161) sixstranded p-strand wrapped around the C-terminal end of a 45 residue extended helix (65 A) that would be part of the pore-forming domain [38]. The receptorbinding function of colicin B was attributed to two antiparallel p-strands (res. 262-282) of the same N-terminal lobe that contains the T-domain. Comparison of the structure of colicin la (Fig. IB) with genetic and functional analysis of the pore-forming colicins implies that the entire coiled-coil might be a good fit to the receptor binding R-domain. Subsequent studies of the minimum receptor binding domain in the A type colicins E9 and E3 that inhibit intracellular protein synthesis have indicated that a large part of the coiled-coil, but perhaps only its distal half, might be necessary for recognition and effective binding to the receptor [18]. The coiled-coil nature of the receptor domain in the structure of intact colicins la and E3 implies that this is a necessary structure motif for interaction with the receptor that initially sequesters the colicin. The markedly smaller length of the extended helices of colicin N and B is suggestive of a mechanism that is different from those used by colicins la and E3 for receptor binding and translocation across the outer membrane and periplasmic space. (D) Cytotoxicity domains. The isolated soluble poreforming 20kDa domain of colicins A [25] and El [10], along with this domain in the intact colicins B, la, and N, have a similar 10-helix globular structure with tightly packed apolar core formed by hydrophobic helical hairpin consisting of helices VIII and IX. The hydrophobic core is isolated from the polar environment by 7-8 hydrophilic helices, part of which have amphipathic patterns. The average length of these helices is 12-13 residues, shorter than required (ca. 20 residues) to span the membrane bilayer. Beyond the implied role of the buried helical hydrophobic hairpin as a potential membrane anchor, the structure of the soluble channel domain provides few clues about the mechanism of pore forming insertion into the membrane. Rather, the structure implies that the soluble C-domain must undergo large conformational changes in order to accomplish the insertion [21, 43]. The function of the hydrophobic helical hairpin is to serve as an anchor during pore-forming domain insertion into membrane bilayer. This interaction
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requires the presence of an optimum content of anionic lipids—that is, an optimum membrane surface potential [44]. Electrostatic interaction with the negatively charged membrane surface (i) guides binding of the C-terminal pore-forming domain with the membrane surface and (ii) induces unfolding that results in the formation of a closed pre-pore state [21, 43]. The ability of pore-forming colicins to form ion channels in planar bilayer membranes was demonstrated for the first time by Schein et al. [29]. The transition of the colicin from a closed-to-open state requires the presence of a transmembrane potential, negative on the side of the membrane opposite to the side of protein binding and insertion. The electrical field across the cytoplasmic membrane (in vivo) created by the electrochemical proton gradient favors poreformation. The structure of the open channel state is not known. The channel is formed by a single colicin molecule [reviewed in [19]]. In the open state of the channel, a significant part of the C-domain (68 residues, helices II-V of colicin la) becomes accessible to ligand binding from the trans side of membrane [16, 33]. The dependence of channel-formation by colicins on the lipid content of membrane points to the involvement of lipids in the formation and structure of ion channel that has been characterized as a "toroidal pore" [42]. Cytotoxic activity of endonuclease colicins also resides in the C-terminal domain. The size of the Cterminal cytotoxic domain of these colicins ranges from 96 to 135 residues, respectively. The immunity proteins bind these domains with very high affinit)^ (Kd < 10"^''M; [39]) efficiently blocking the interaction of colicins with their substrates, DNA or RNA. Extensive, highly complementary interaction surfaces in endonucleaseImm complexes that involve hydrophobic and polar side chains of both proteins, account for the highaffinity nature of Imm binding. Active sites in both DNase and rRNase colicins involve residues C terminal to the residues involved in Imm binding. This can allow Imm binding to an incompletely translated colicin in order to afford protection to host cells during the toxin synthesis.
MECHANISM OF IMPORT ACROSS OUTER MEMBRANE Colicins parasitize receptors that have a metabolic function in the binding and transport of vitamins, metals, and sugars. The best characterized colicin receptor system is that of vitamin B12 receptor (BtuB) utilized by colicin E3 (Table 1). The colicin binds tightly (Ka < 10"^^ M) to BtuB. A 2.75 A structure of BtuB-R-domain has been obtained [18]. Translocation of colicin E3
across the outer membrane appears to require a second outer membrane protein, the OmpF porin [2], to which the T-domain can bind, forming a two-receptor translocon for colicin import across the outer membrane. The coupled and concomitant events required for this import are (i) high-affinity binding to BtuB [37], (ii) unfolding of the colicin and extracellular release of immunity protein, (iii) insertion of disordered Nterminal region of the T-domain into the OmpF channel [41], and (iv) presumed cleavage of C-domain at an exposed R-C junction [9, 32].
AREAS OF RESEARCH ENCOMPASSED BY COLICIN STUDIES As just described, colicin studies span a range of general interests that include mechanisms of protein secretion, protein import, protein-receptor interactions, high-affinity protein-protein interactions, ion channel structures and function, and intracellular nuclease activities. Acknowledgment Studies of the authors reported here were supported by a grant (GM-18457) from the NIH.
References [1] Almendinger R, Hager LP. Studies on the mechanism of action of colicin E2, 107-33. In "Chemistry and function of colicins" (Hager LP, ed.). Academic Press, New York. [2] Benedetti H, Frenette M, Baty D, Lloubles R, Geli V, Lazdunski C. Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coll J Gen Microbiol 1989;135:3413-20. [3] Bishop LJ, Bjes ES, Davidson VL, Cramer WA. Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin El. J Bacteriol 1985;164:237-44. [4] Bouveret E, Rigal A, Lazdunski C, Benedetti H. The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step. Mol Microbiol 1997;23:909-20. [5] Bowman CM, Sidikaro J, Nomura M. Specific inactivation of ribosomes by colicin E3 in vitro and mechanism of immunity in colicinogenic cells. Nat New Biol 1971;234:133-7. [6] Braun V, Patzer SI, Hantke K. Ton-dependent colicins and microcins: modular design and evolution. Biochimie 2002;84: 365-80. [7] CardelliJ, KoniskyJ. Isolation and characterization of an Escherichia coli mutant tolerant to colicins la and lb. J Bacteriol 1974;119:379-85. [8] Cavard D, Oudega B. (1992). General introduction to the secretion of bacteriocins, 297-305. In "Bacteriocins, Microcins, and Lantibiotics" (James R, Lazdunski C, Pattus F, eds.). SpringerVerlag, Berlin. [9] de Zamaroczy M, Mora L, Lecuyer A, Geli V, Buckingham RH. Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB. Mol Cell 2001;8:159-68.
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES [10] Elkins P, Bunker A, Cramer WA, Stauffacher CV. A mechanism for protein insertion into the membranes is suggested by the crystal structure of the channel-forming domain of colicin El. Structure 1997;5:443-58. [11] Geli V, Baty D, Pattus F, Lazdunski C. Topology and function of the integral membrane protein conferring immunity to colicin A. Mol Microbiol 1989;3:679-87. [12] Gould JM, Cramer WA. Studies on the depolarization of the Escherichia coli cell membrane by colicin El. J Biol Chem 1977;252:5491-97. [13] Graille M, Mora L, Buckingham RH, van Tilbeurgh H, de Zamaroczy M. Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein. EMBO J 2004;23: 1474-82. [14] Hilsenbeck JL, Park H, Chen G, Youn B, Postle K, Rang C. Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution. Mol Microbiol 2004;51:711-20. [15] Jakes R, Zinder ND. Highly purified colicin E3 contains immunity protein. Proc Natl Acad Sci USA 1974;71:3380-84. [16] Rienker PR, Jakes R, Blaustein RO, Miller C, Finkelstein A. Sizing the protein translocation pathway of colicin la channels. J Gen Physiol 2003;122:161-76. [17] Riihlmann UC, Pommer AJ, Moore GR, James R, Rleanthous C. Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes. J Mol Bio 2000;301:1163-78. [18] Rurisu G, Zakharov SD, Zhalnina MV, Bano S, Eroukova VY, Rokitskaya TI, Antonenko YN, Wiener MC, Cramer WA. The structure of BtuB with bound colicin E3 R-domain implies a translocon. Nat Struct Biol 2003;10:948-54. [19] LakeyJH, Slatin SL. Pore-forming colicins and their relatives. Curr Top Microbiol Immunol 2001;257:131-61. [20] Lazzaroni J-C, Dubuisson J-F, Vianney A. The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins. Biochimie 2002;84:391-97. [21] Lindeberg M, Zakharov SD, Cramer WA. Unfolding pathway of the colicin El channel protein on a membrane surface. J Mol Biol 2000;295:679-92. [22] Luria SE. On the mechanisms of action of colicins. Annales de L'Institut Pasteur 1964;107:67-73. [23] Nomura M. Mechanism of action of colicins. Proc Natl Acad Sci USA 1964;52:1514-21. [24] Ohno-Iwashita Y, Imahori R. Assignment of the functional loci in colicin E2 and E3 molecules by the characterization of their proteolytic fragments. Biochemistry 1980;19:652-59. [25] Parker MW, Postma JPM, Pattus F, Tucker AD, Tsernoglou D. Refined structure of the pore-forming domain of colicin A at 2.4 A resolution. J Mol Biol 1992;224:639-57. [26] Phillips SR, Cramer WA. Properties of the fluorescence probe response associated with the transmission mechanism of colicin El. Biochemistry 1973;12:1170-76. [27] Postle R, Radner RJ. Touch and go: tying TonB to transport. Mol Microbiol 2003;49:869-82. [28] Reeves P, ed. (1972). "The Bacteriocins," Chapter 1. SpringerVerlag, New York.
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[29] Schein SJ, Ragan BL, Finkelstein A. Colicin R acts by forming voltage-dependent channels in phospholipid bilayer membranes. Nature 1978;276:159-63. [30] Schneider CG, Penfold CN, Moore GR, Rleanthous C, James R. Identification of residues in the putative TolA box which are essential for the toxicity of the endonuclease toxin colicin E9. Microbiology 1997;143:2931-38. [31] Schramm E, MendeJ, Braun V, Ramp RM. Nucleotide sequence of the colicin B activity gene cba: Consensus pentapeptide among TonB-dependent colicins and receptors. J Bacteriol 1987;169:3350-57. [32] Shi Z, Chak RF, Yuan HS. Identification of an essential cleavage site in ColE7 required for import and killing of cells. J Biol Chem 2005;280:24663-8. [33] Slatin SL, Qiu X-Q, Jakes RS, Finkelstein A. Identification of a translocated protein segment in a voltage-dependent channel. Nature 1994;371:158-61. [34] SmardaJ. (1978). "The effects of colicins," p. 213. J. E. Purkyne University Brno. [35] Soelaiman S, Jakes R, Wu N, Li CM, Shoham M. Crystal structure of colicin E3: Implications for cell entry and ribosome inactivation. Mol Cell 2001;8:1053-62. [36] Sui MJ, Tsai LC, Hsia RC, Doudeva LG, Ru WY, Han GW, Yuan HS. Metal ions and phosphate binding in the H-N-H motif: crystal structures of the nuclease domain of Cole7/Im7 in complex with a phosphate ion and different divalent metal ions. Prot Sci 2002;11:2947-57. [37] Taylor R, Burgner JW, Clifton J, Cramer WA. Purification and characterization of monomeric Escherichia co/? vitamin B12 receptor with high affinity for colicin E3. J Biol Chem 1998;273:31113-18. [38] Vetter IR, Parker MW, Tucker AD, Lakey JH, Pattus F, Tsernoglou D. Crystal structure of a colicin N fragment suggests a model for toxicity. Structure 1998;6:863-74. [39] Walker D, Moore GR, James R, Rleanthous C. Thermodynamic consequences of bipartite immunity protein binding to the ribosomal ribonuclease colicin E3. Biochemistry 2003;42:4161-71. [40] Wiener M, Freymann D, Ghosh P, Stroud RM. Crystal structure of colicin la. Nature 1997;385:461-64. [41 ] Zakharov SD, Eroukova VY, Rokitskaya TI, Zhalnina MV, Sharma O, Loll PJ, Zgurskaya HI, Antonenko YN, Cramer WA. Colicin occlusion of OmpF and TolC channels: outer membrane translocons for colicin import. Biophys J 2004;87:3901-11. [42] Zakharov SD, Rotova EA, Antonenko YN, Cramer WA. On the role of lipid in colicin pore formation. Biochim Biophys Acta 2004;1666:239-49. [43] Zakharov SD, Lindeberg M, Griko YV, Salamon Z, Tollin G, Prendergast FG, Cramer WA. Membrane-bound state of the colicin El channel domain as an extended two-dimensional helical array. Proc Nad Acad Sci USA 1998;95:4282-87. [44] Zakharov SD, Rokitskaya TI, Shapovalov VL, Antonenko YN, Cramer WA. Tuning the membrane surface potential for efficient toxin import. Proc Nad Acad Sci USA 2002;99:8654-59. [45] Zhang YL, Cramer WA. Intramembrane helix-helix interactions as the basis of inhibition of the colicin El ion channel by its immunity protein. J Biol Chem 1993;268:10176-84.
Colicins: Bacterial/Antibiotic Peptides O. SHARMA, S. D. ZAKHAROV, AND W. A. CRAMER
tor specific for that type of colicin. Indeed, as was confirmed later, colicins enter cells by parasitizing receptors in the outer membrane whose physiological purpose is the binding and import of metabolites (e.g., vitamins, sugars, metals such as iron). Subsequently, several colicins differing in their mode of action (discussed in [34]) and the receptors they parasitize to gain entry into target cells, have been identified. Ribbon diagrams of those colicins (la, N, B, which are pore formers and E3, an rRNase), whose 3-D structure has been solved, are shown in Fig. 1. Colicins have been classified according to the translocation system that they appropriate to enter the bacteria. Group A colicins utilize the bacterial Tol-dependent translocation system consisting of the proteins TolA, TolB, TolQ, TolR, and Pal (Table 1). The name of these proteins is derived from the fact that their mutants are "tolerant" to colicin action. Group B colicins utilize the Ton-dependent system, consisting of TonB and ExbB-ExbD (Table 2). "Ton" mutants are resistant to the phage Tl—hence the name "Ton." As can be seen from the tables, the lethal action of most colicins is exerted either by cellular de-energization [22, 26] that results from the formation of a highly conducting pore [29] in the cytoplasmic membrane [12], or by an enzymatic nuclease digestion mechanism [1, 23, 24].
ABSTRACT The functions of receptor binding, cytotoxicity, and translocation across the cell envelope, necessary for colicins to be imported into the cell and to exert their lethal effect on the cell, are encoded in defined peptide domains of the colicin polypeptide. The major lethal effects of the colicins are exerted through pore formation and intracellular nuclease activity. High-resolution x-ray structures of four colicins and x-ray/NMR structures of four C-terminal domains encoding cytotoxic function are known. Cells producing colicin are protected against autocytotoxicity by a -lOkDa immunity protein, which is a tightly bound soluble component of the nuclease colicins or a membrane-bound peptide present in low abundance in cells producing poreforming colicins. Import into the cell is guided by a transenvelope network of "tol" or "ton" proteins. DISCOVERY AND CLASSIFICATION [28] Colicins are plasmid-encoded bacteriocins, produced hy Escherichia co/z under stress conditions, which are cytotoxic to closely related strains that contain the required outer membrane receptor(s) but do not produce the cognate immunity protein. Colicins were first discovered in 1925 by Gratia who showed that one strain oi E. coli ("V" for virulent) released a substance found in the filtrate that was toxic to a sensitive strain. Gratia also showed that colicin-resistant mutants could be derived from sensitive strains. Fredericq later suggested that resistance to colicins involved the loss of a surface recepHandbook of Biologically Active Peptides
STRUCTURE OF THE PRECURSOR mRNA/GENE Colicin-producing cells are protected from its cytotoxic action by coordinate synthesis of an immunity 115
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TABLE 1. Colicins that are imported via the Tol system. Although these colicins have a common theme of import into target cells, the receptors and translocators utilized by these colicins are different. They also differ in their cytotoxic activities. Colicin A^ E1 N E2 E7 E9 E3 E6
Activity Protein^
Immunity Protein^
Receptor
Translocation
Cytotoxic Activity
592 522 387 581 566 582 551 551
178 113 131 86 87 86 85 85
BtuB BtuB OmpF BtuB BtuB BtuB BtuB BtuB
OmpF, TolQRAB TolC, TolQRAB TolQRA OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB OmpF, TolQRAB
Pore forming Pore forming Pore forming DNase DNase DNase 16SrRNase 16SrRNase
^Length In amino acids. ^Sequence of Colicin A from Citrobacter freundii. Tol-dependent colicins E4, E5, E8, K, U, 28b, and DF13 have not been included in the table. Modified from [20].
TABLE 2. Colicins that are imported via the Ton system. Although the mechanism of import of these colicins into the target cells are similar, the cytotoxic activity differs. A common feature of these colicins is the presence of a TonB box that interacts with the TonB protein which transduces energy from the inner membrane to these import processes [27]. Activity Protein^
Immunity Protein^
Receptor protein
B D
510 697
175 87
FepA FepA
la lb
626 626
111 115
CIr Cir
Colicin
Activity Channel Protein synthesis^ Channel Channel
^Length in amino acids. ^Inhibits protein synthesis by cleavage of arginine tRNAs. Ton-dependent colicins Js, M, 5 and 10 have not been included in the table. Modified from [6].
protein that acts specifically against that colicin. Immunity proteins are small (MW -lOkDa) polypeptides that protect the colicin-producing cells from both endogenous and exogenous colicins [5,15]. In the case of pore forming colicins, a small number of immunity molecules (-10^-10^) in the cytoplasmic membrane are able to provide immunity against a functional and lethal channel in that membrane [45]. Regions of homology between the immunity proteins of pore-forming colicins A, B and N have suggested that extramembrane loops LI and L3 of the 3-4 TM helix protein interact with the respective colicins. Thus, mutations in these regions affect the immunity activity of these proteins [11]. A lysis protein localized in the periplasmic space functions to release colicin from producing cells to the extracellular medium, both for colicin-immunity
protein complex of enzymatically active colicins and for pore-forming colicins. In pore-forming colicins, the Imm protein remains in the cytoplasmic membrane [3]. The gene encoding colicin, its cognate immunity protein, and the lysis protein form an operon that is regulated by the SOS (DNA damage-induced) promoter [8]. In the absence of induction, the immunity gene is expressed by a constitutive promoter that provides basal levels of immunity protein for protection against exogenous colicins. THE DOMAIN CONCEPT There are three defined steps in the killing of a sensitive E. coli by most colicins, each of which is carried
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES
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A: H2N
COOH
FIGURE 1. (A) Generic domain structure of colicin polypeptides (top): T, translocation; R, receptor binding; C, catalytic or activity containing domain (B-E). Ribbon diagrams of structures of pore-forming colicins, la (B), B (E), and N (D), and of rRNase colicin E3 (C). Note the prominent long (210 A and 100 A in colicins la and E3) coiled-coil structure of the R-domain. Structures, predominantly a-helical, have also been obtained of the C-domains of (i) colicins A and El by x-ray diffraction, and (ii) of E7 and E9 by solution NMR. The structures of E3 and E7 contain bound immunity protein, as is characteristic of the nuclease colicins. (See color plate.)
out by a separate domain of the colicin that occupies approximately one-third of the polypeptide (Fig. lA; [24]). The first step involves the central "R" (receptor) domain, which mediates the binding of colicin to its outer membrane receptor. The N-terminal "T" (translocation) domain mediates the second step of translocation from the outer membrane receptor to the colicin target in the cell. Finally, the carboxy-terminal "C" (catalytic) domain functions in cytotoxicity. SEQUENCE COMPARISONS (Fig. 2) Groups A and B have subgroups with extended regions of high homology. In group A, colicins with endonuclease activity (E2-E9) have a high degree of homology in the T and R domains (residues 1-449) based on 85% similarity (70% for E7). The subgroup of pore-forming colicins (El, A, and N) have low homol-
ogy between thvemselves and with colicins E2-9 (-13% between any two pore-forming group A colicins) in the T and R domains. However, the homology is higher between C-domains of pore-forming colicins (-30%), which correlates with a common motif consisting of a 10-helix globular structure including a hydrophobic helical hairpin. The subgroup of endonuclease colicins has high homology in the C-domain between IGsRNase colicins E3 and E6 (90%), and between DNase colicins E2, E 7 a n d E 9 (80%). A conserved pentapeptide sequence "TolB box" is present in most of the Tol-dependent colicins. This TolB box has been shown to interact with the TolB protein [4, 30], defining a step in the translocation process. A site of proteolytic cleavage between TR- and C-domains (R447) has been defined in E7 [32]. In group B, the pore-forming colicins la and lb use the CirA iron transport receptor for recognition [6, 7]. They are identical in the T and R domains including
Group A
E7 : E3 : E6 : E2 : E9 : El: A: N:
1 21 41 61 i l l I MSGGDGRGHNSGAHNTGGNI NGGPTGLGGNGGASDGSGWSSENNPWGGGS GSGVHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGLGVGGGASDGSGWSSENNPWGGGS GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGLGVGGGASDGSGWSSENNPWGGGS GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGLGVGGGASDGSGWSSENNPWGGGS GSGIHWGGGSG MSGGDGRGHNTGAHSTSGNI NGGPTGIGVSGGASDGSGWSSENNPWGGGS GSGIHWGGGSG METAVAYYKDGVPYDDKGQVIITLLNGTPDGSGSGGGGGKGGSKSESSAAI HATAKWSTAQL -MPGFNYGGKG DGTGWSSERGSGPEPGGGSHGNSGGHDRGDSSNVGNESVTVMKPGDSYNTPVv^GKV MGSNG ADNAHNNAFGGGKNPGIGNTSGAGSNGSASSNR GNSNGWSWSNK
E7 : E3 : E6 : E2 : E9 : El: A: N:
81 101 121 I I I HGNGGGNSNSGGGS NSSVAAPMAFG- -FPALAAPGAGTLGISVSGEALSAAIADIFAALKGPFKFSAWGIALYGI HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG- -FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGV HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG- -FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA HGNGGGNGNSGGGSGTGGNLSAVAAPVAFG--FPALSTPGAGGLAVSISAGALSAAIADIMAALKGPFKFGLWGVALYGA KKTQAEQAARAKAAAEAQAKAKANRDALTQRLKDIVNEALMQAEDE-RLRLAK AEEKARKEAEAAR IINAAGQPTMNGTVMTADNSSMVPYGRGFTRVLNSLVNNPLMVQSG-NLPPGYIAJLSNGKVMTEV REERTSGGGGKNV PHKNDGFHSDG SYHITFHGDNNSKPKPGTITPDN G
E7 : E3 : E6: E2 : E9 : El: A: N:
141 161 181 201 I I I I LPSEIAKDDPNMMSKIVTSLPAETVTNVQVSTLPLDQATVSVTKRV TDWKDTRQHIAWAGVPMSVPWNAKPTR LPSQIAKDDPNMMSKIVTSLPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE GGLAVSISAGALSAAIADILPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE GGLAVSISAGALSAAIADILPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE GGLAVS ISASELSAAIAGILPADDITESPVSSLPLDKATVNVNVRV VDDVKDERQNISWSGVPMSVPWDAKPTE HNASRTPSATELAHANNAAEKAFQEAEQRRKEIEREKAETERQLKL AEAEEKRLAALSEEAKAVEIAQKKLSA SPAGQNGGKSPVQTAVENYGNERTWTVKVPREVPQLTASYNEGMRIRQEAADRARAEANARALAEEEARAIASGKSKAEF NSGNRGNNGDGASAKVGEI S
E7: E3: E6: E2: E9: El: A: N:
221 241 261 281 I I I I TPGVFHASFPGVPSLTVSTVKGLPVSTTLPRGITEDKGRTAVPAGFTFGGGSHEAVIRFPKESGQKPVYVSVTDVLTPAQ RPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDQ RPGVFTASIPGAPVLNISVNNSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP RPGVFTASIPGAPVLNISVNNSTPEVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP RPGVFTASIPGAPVLNISVNDSTPAVQTLSPGVTNNTDKDVRPAGFTQGGNTRDAVIRFPKDSGHNAVYVSVSDVLSPDP AQSE V^/KMDGEIKTLNSRLSSSI--HARDAEMKTLAGKRISIELAQAIREEKQKQVTASETRINRINS DAGKRVEAAQAA INTAQLIWNNLSGAVSAAN- -QVITQKQAEMTPLKNELAAAJST^^DTKQNEINAAVANRDALNN KPGR YISSNPEYSLLAKLIDAES P DSNIDKMRVDYVN-
E7 : E3 : E6 : E2 : E9 : El: A: N:
301 321 341 361 I I I I VKQRQDEEKRLQQEWNDAHPVEVAERNYEQARAELNQANKDVARNQERQAKAVQVYNSRKSELDAAN VKQRQDEENRRQQEWDATHPVEAAERNYERARAELNQANEDVARNQERQAKAVQVYNSRKSELDAAN KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN KDSGHNAVYVSVSDVLSPDQVKQRQDEENRRQQEWDATHEDVARNQERQAKAVQVYNSRKSELDAAN AKYKELDELVKKLSPRANDPLQNRPFFEATRRR-VGAGKADITQIQKAISQVSNNRNAGIARVHEAE QRVQETLKFI NDPIRSRIHFNMRSGL- IRAQHSQLSQAmJILQNARNEKSAADAALSAATAQRLQAEAALRAA - - IKGTEVYT FHTRKGQYVKVTVWKGPKYNNKLVK RFVSQFLLFRKE
E7 E3 E6 E2 E9 El
381 401 I I KTLADAKAEI —KQFERFAREPMAAGHRMWQMAGLKAQRAQTDVNNKKAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE KTLADAIAEI - -KQFNRFAHDPMAGGHRMWQMAGLKAQRAQTDVNNKQAAF-DAAAKE ENLKKAQI^nSTLLNSQIKDAVDATVSFYQTLTEKYGEKYSKMAQELADKSK GKKIGN FIGURE 2, Amino acid residues of colicins E3 and la are numbered in groups A and B, respectively. Residues considered to be similar are: D and E; K and R; N and Q; S and T; I, L, and V. Coloring: red (bold)—similar residues In all or most (except one) of sequences. Blue (bold)—similar residues in poreforming colicins (E1, A, N) of group A. Orange and green—used to show similarity within subgroups of DNase and RNase colicins of group A. Green and blue—used to show similarity within subgroups of CirA- and FepAdependent colicins of group B. Alignment was done using the MUSCLE program.
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES A: N-
/
AEAAEKARQRQAEEAERQRQAMEVAEKAKDER- - ELLEKTSELIAGMGDKIGEHLGDKYKAIAKDIADNIKNPQGKTIRS EKEKNEK- -EALLKASELVSGMGDKLGEYLGVKYKJSrVA^^^^ 421 I
E7: E3 : E6 : E2: E9: El: A: N:
441 I
461 I
KSDADVALSSALERRKQKENK-EKDAKAKLDKESKRNKPGKATGKGKPVlSnsrKWLNNAGKDLGSPVPDRIANKL KSDADAALSSAMESRKKKEDK-KRSAENNLNDEKNKPRKG FKDYGHDY-HPAP KTENIKG KSDADAALSSAMESRKKKEDK-KRSAENKLNEEKNKPRKG VKDYGHDY-HPDP KTEDIKG KSDADAALSAAQERRKQKENK-EKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKN KSDADAALSAAQERRKQKENK-EKDAKDKLDKESKRNKPGKATGKGKPVGDKWLDDAGKDSGAPIPDRIADKLRDKEFKS WTEALAAFEKYKDVLNKKFSK/iDRDAiraALASVKYDDWAKH LDQFAKYLKITGHVSFGYDWSDILKI FDDA^mSLNKITM^IPAI4KIOTCADRDAL^/HAWKHVDAQD^lA^IK LGNLSKAFKVADWMKVEKVREKSIEG YNEAJ^ASLNKVLANPKt^KVNKSDKDAIVNAWKQVNAKDMANK IGNLGKA,FKVADLAIKVEKIREKSIEG 481 I
501 I
521 I
E7 : E3 : E6: E2 : E9: El : A: N:
F — DDFRKKFWEEVSKDPELSKQFSRNNNDRMKVGKA PKTRTQDVSGKRTSFELHHEKPISQNGGVYDMDNISWT L--GDLKPGI PKTPKQNGGGKRKRWT GDKGRKIYEWDSQHGELEGYRASDGQHLGSFD L--GELKEGK PKTPKQGGGGKRARWY GDKGRKIYEWDSQHGELEGYRASDGQHLGSFE F--DDFRKKFWEEVSKDPDLSKQFKGSNKTNIQKGKA PFARKKDQVGGRERFELHHDKPISQDGGVYDJyOSINIRVTT F--DDFRKAVWEEVSKDPELSKNLNPSNKSSVSKGYS PFTPKNQQVGGRKVYELHHDKPISQGGEVYDMDNIRVTT KDTGDWKPLF LTLEKKAADAGVSYWALLF5S LLAGTTLGIWGIATVTGILCSYIDKNKLNTIN YETG^JWGPLM LEVESIWLSGIASSVALGIFSATLGAYALSLGVPAIAVGIAGI -LLAA\7VGALIDDKFADALN YNTGNWGPLL LEVESVfl IGGVVAGVAISLFGAVLSFLPIS -GLAVTALGVIGI -MTISYLSSFIDANRVSNIN
E7: E3: E6: E2: E9:
541 I PKRHIDIHRGK PKTGNQLKGPDPKRNIKKYL PKTGNQLKGPDPKRNIKKYL PKRHIDIHRGK PKRHIDIHRGK
El: A: N:
EVLGI NEIIRPAH NIISSVIR
la: lb: B: D:
1 21 41 61 I I I I MSD PVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIWGGNEVvVDDSPTRSDIEKRDK MSD PVRITNPGAESLGYDSDGHEIMAVDIYVNPPRVDVFHGTPPAWSSFGNKTIVvGGNEV\IVDDSPTRSDIEKRDK MSDNEGSVPTEGIDYGDTMWWPSTGR-IPGGDVKPGGSS-GLAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKEL MSDYEGSGPTEGIDYGHSMWWPSTGL-ISGGDVKPGGSS-GIAPSMPPGWGDYSPQGIALVQSVLFPGIIRRIILDKEL 81 I
Group B
101 I
121 I
141 I
la: lb: B: D:
EITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADITRQEFRLLQAELREYGFRTEIAGYDA EITAYKNTLSAQQKENENKRTEAGKRLSAAIAAREKDENTLKTLRAGNADAADITRQEFRLLQAELREYGFRTEIAGYDA EEGDWSGWSVSVHSPWGNEKVSAARTVLE NGLRGGLPEPSRPAAVSF EEGDWSGWSVSVHSPWGNEKVSAARTVLE NGLRGGLPEPSRPAAVSF
la: lb: B: D.
161 181 201 221 I I I I LRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQNADKKAA--DMLAEyERRKGILDTRLSELEKNGGAALAVLD LRLHTESRMLFADADSLRISPREARSLIEQAEKRQKDAQMADKKAA--DMLAEYERRKGILDTRLSELEKNGGAALAVLD ARLEPASGNEQKI IRLMVTQQLEQVTDIPASQLPAAGNNVPVKY RLTDLMQNGTQYMAIIG ARLEPASGNEQKI IRLMVTQQLEQVTDI PASQLPAAGNNVPVKY RLMDLMQNGTQ YMAIIG
la: lb: B: D:
241 261 281 301 I I I I AQQARLLGQQTRNDRAISEARNKLSSVTESLNTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIWSGDPR AQQARLLGQQTRNDRAISEARNKLSSVTESLKTARNALTRAEQQLTQQKNTPDGKTIVSPEKFPGRSSTNHSIV\;SGDPR GIPMTV PWDAVPVPDR--SRPGTNIKDVYSAPVSPNL--PDLVLSVGQ!4NTPVRSNPEIQ GIPMTV PWDAVPVPDR- - SRPGTNIKDVYSAPVSPNL- -PDLVLSVGQMNTPVLSNPEIQ 321
FIGURE 2. (Continued)
341
361
381
119
120
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CHAPTER 18
la: lb: B: D:
I
I
401 la: lb: B: D:
I
441 I
421
AVNSARNNLS ARTNE QKHANDALNALLKEK ENIRNQLSGINQKIAEEKRKQDELKATKDAI - N F AINSARNIWSARTNE QKHANDALNALLKEK E.NIRSQLADINQKI AEEKRKRDEINMVPCDAI -KL AENNAKDDFRVKKEQ ENDEKTVL . TKTSEVIISVGDKVGEY AENKZOCDDFRVKKEEAVARAEAEKAKAELFSKAGVNQPPVYTQEMMERANSVMNEQGALVLNNTASSVQLAM^^^ 461 I
la: lb: B: D:
481 I
501 I
TTEFLKSVSEKYGAKAEQLARE]yL2iG QAKGKKI RI^TVEEALKTYEKYRADIN TSDFYRTIYDEFGKQASELAKELAS VSQGKQI KSVDDALNAFDKFRNNLN LGDKYKALSREIAENINNF QGKTI RSYDDAMSSINKLMANPS AGDIAGNISKFFSNALEKVTIPEVSPLLMRISLGALWFHSEEAGAGSDIVPGm^LEAMFSLSAQMLAGQGWIEPGATSV 521
la: lb: B: D:
I
FAGTIKITTSAVIDNRANLNYLLSHSGLrJYKRNILNDRNPVVT-EDVEGDKK FAGTIKITTSAVIDNR.2U^JL^^yLLTHSGLDYKRNILNDRNPVVT-EDVEGDKKIYNAE:VAEWDKLRQRLLDARNKITSAE5^ EDGVISET GNYVEAGYTMSS NNHDVIVRFPEGSGVSPLYISAVE ILDS-NSLSQRQE EEGVIAET GNYVEAGYTMSS NNHDVIVRFPEGSDVSPLYISTVE ILDS -NGLSQRQE
541
I
561
KKINAKDRAAIAAALESVKLSDISSNLNRFSRGL6YAGKFTSLADWITEFGKAVRTENWR.P KKYNIQDRMAISKALEAINQVHMAENFKLFSKAFGFTGKVIERYDVAVELQKAVKTDNWRP LKINATDKEAIVNAWKAFNAEDMGNKFAALGKTFKAADYAIKANNIREKSIEGYQTGNWGP NLPVRGQLINSNGQLALDLLKTGNESIPAAVPVLNAVRDTATGLDKITLPAV^/GAPSRTILVNPVPQPSVPT-DTGNHQP 581
I
la: lb: B: D:
LEV KTETII AGN/\ATALVALVFSILTGSA FPV KLESLA AGRAASAVTAWAFSVMLGTP LML EVES V^A/--ISGMASAVALSLFSLTLGSALIA EGLS VPVTPVHTGTEVKSVEMPVTTITPVSDVGGLRDFIYVJRPDAAGTGVEAVYVMLNDPLDSGRFSRKQLDKKYKHAGDFGIS 601
621
la: LGIIGY GLLIvmVTGALIDESLV--EKANKFWG 1 lb: VGILGF AIIMAAVSALVNDKFI--EQVNKLIG 1 B: ATWGF VGWIAGAIGAFIDDKFV--DELNHKII K D: DTKKNRETLTKFRDAIEEHLSDKDTVEKGTYRREKGSKVYFNPNTMNWIIKSNGEFLSGWKINPDADNGRIYLETGEL
FIGURE 2. (Continued)
most of the coiled-coil structure (residues 1-426), and have a 60% similarity in the C-domain. CoHcins B and D containing 510 and 627 residues, respectively, act through pore-forming and DNase activities, and use the ferro-enterobactin receptor FepA for recognition [6]. They have a high degree of homology (95%) in the Nterminal segment (res. 1-310). The homology of the aligned segment of central R- and C-terminal domains, excluding that unique to colicin D, is smaller (-30%). Homology between any three of the four colicins of group B aligned in Fig. 2 is 25%. A common feature of the Ton-dependent colicins is the presence of a "TonB box" pentapeptide sequence in a position proximal to the N-terminus [31]. This sequence is present not only in the Ton-dependent colicins, but also in the outer membrane receptor proteins whose function is dependent on the Ton system.
X-RAY STRUCTURES OF COLICINS (Figs. IB-E) (A) The four complete or incomplete structures of colicins that have been solved by x-ray diffraction of three-dimensional crystals and their major properties are summarized: (i) The structure of residues 23-39 and 83-624 of the 626 residue pore-forming colicin la ("B-type," translocated by the Ton system) has been solved to 3.0 A resolution (Fig. IB; [40]). (ii) The structure encompassing residues 84-551 of the 551 residue "A-type" (translocated by the Tol system) endoribonuclease colicin E3 has been solved to 3.0 A (Fig. IC; [35]). The x-ray structure of a complex of the BtuB receptor with bound coiled-coil R-domain of colicin E3 [18], which is relevant to the mechanisms of cellular import, is discussed below, (iii) 292 residues, 93-385,
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES
of a 321 residue chymotryptic fragment of the 387 residue pore-forming colicin N, has provided a 3.1 A structure that contains the globular channel forming domain (C, in blue, Fig. ID) but is missing a substantial part of the T- (and possibly of the R-) domain [38]. (iv) The structure of most of the 511 residue poreforming colicin B (Ton-dependent) (Fig. IE) was solved at 2.5 A resolution [14]. The x-ray and NMR structures of the C-domains of pore-forming colicins A [25] and El [10] show a bundle of 10 a-helices with a hydrophobic helical hairpin anchor at the core. Structures of the endonuclease domain of colicins E3, E7, E9 (Toldependent), and Ton-dependent colicin D in complex with Imm or nucleic acids have also been solved [13, 17, 36]. The T-, R-, and C-domains in colicins E3 (RNase; Tol-dependent) and la (pore-forming; Ton-dependent) are structurally defined (Figs. IB, C) and connected by long a-helices that form coiled-coils, whereas in colicins B (pore-forming; Ton-dependent; Fig. IE), and probably in N (pore-forming; Tol-dependent; Fig. ID), the T- and R-domain functions are located in a globular domain which is separated from the C-domain by a single long helix, forming the shape of a dumbbell. These two distinct shapes are not related to either the import pathway (Tol- or Ton-dependent), or to the mechanism of cytotoxicity (pore-formation or endonuclease). It has been proposed that the coiled-coil structure in colicin E3 is necessary for delivery of T- and C-domains of a receptor-bound colicin to an outer membrane porin through which the C-domain, at least, is translocated to the periplasm [18]. However, involvement of outer membrane porins in the import of Tondependent colicin la has not yet been implied. (B) T-domains. The N-terminal T-domain of colicin la contains a "TonB box" (E23-V27) that is necessary for translocation across the periplasm as part of an antiparallel helix bundle. This secondary structure of the T domain of colicin la contrasts with the p-sheet "jelly roll" that forms most of the colicin E3 T domain. The N-terminal region of the colicin E3 T domain contains a DGSGW (D35-W39) "TolB box" needed for translocation [4] and is glycine-rich (34 of 79 residues), accounting for the disorder in this region of the structure. A possible reason for the difference in the Tdomain structures of colicins la and E3 is that the immunity protein is tightly bound to the T domain in the latter. The N-terminal domain of colicin B is composed of 17 P-strands, 2 a-helices, and long loops. The central region (residues 130-291) of its sequence shares some similarity with the T domain of colicin E3, but the folding pattern does not fit to a jellyroll motif. In both colicins la and B, the N-terminal segments, containing the TonB box (residues 23-27 and 17-21 in la and B, respectively) are folded back and interact with antipar-
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121
allel helices of the coiled-coil and T-domain in colicin la, and with the N-terminal lobe (TR-domains) of colicin B. (C) R-domains. The 104 residue region at the tip of the 160 A coiled-coil, consisting of an amphipathic twostranded P-hairpin folded around the tip-terminal helix of the coiled-coil was proposed to bind to the colicin la Cir receptor [40]. The R-domain of colicin N was proposed to consist of a 65 residue (residues 97-161) sixstranded p-strand wrapped around the C-terminal end of a 45 residue extended helix (65 A) that would be part of the pore-forming domain [38]. The receptorbinding function of colicin B was attributed to two antiparallel p-strands (res. 262-282) of the same N-terminal lobe that contains the T-domain. Comparison of the structure of colicin la (Fig. IB) with genetic and functional analysis of the pore-forming colicins implies that the entire coiled-coil might be a good fit to the receptor binding R-domain. Subsequent studies of the minimum receptor binding domain in the A type colicins E9 and E3 that inhibit intracellular protein synthesis have indicated that a large part of the coiled-coil, but perhaps only its distal half, might be necessary for recognition and effective binding to the receptor [18]. The coiled-coil nature of the receptor domain in the structure of intact colicins la and E3 implies that this is a necessary structure motif for interaction with the receptor that initially sequesters the colicin. The markedly smaller length of the extended helices of colicin N and B is suggestive of a mechanism that is different from those used by colicins la and E3 for receptor binding and translocation across the outer membrane and periplasmic space. (D) Cytotoxicity domains. The isolated soluble poreforming 20kDa domain of colicins A [25] and El [10], along with this domain in the intact colicins B, la, and N, have a similar 10-helix globular structure with tightly packed apolar core formed by hydrophobic helical hairpin consisting of helices VIII and IX. The hydrophobic core is isolated from the polar environment by 7-8 hydrophilic helices, part of which have amphipathic patterns. The average length of these helices is 12-13 residues, shorter than required (ca. 20 residues) to span the membrane bilayer. Beyond the implied role of the buried helical hydrophobic hairpin as a potential membrane anchor, the structure of the soluble channel domain provides few clues about the mechanism of pore forming insertion into the membrane. Rather, the structure implies that the soluble C-domain must undergo large conformational changes in order to accomplish the insertion [21, 43]. The function of the hydrophobic helical hairpin is to serve as an anchor during pore-forming domain insertion into membrane bilayer. This interaction
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CHAPTER 18
requires the presence of an optimum content of anionic lipids—that is, an optimum membrane surface potential [44]. Electrostatic interaction with the negatively charged membrane surface (i) guides binding of the C-terminal pore-forming domain with the membrane surface and (ii) induces unfolding that results in the formation of a closed pre-pore state [21, 43]. The ability of pore-forming colicins to form ion channels in planar bilayer membranes was demonstrated for the first time by Schein et al. [29]. The transition of the colicin from a closed-to-open state requires the presence of a transmembrane potential, negative on the side of the membrane opposite to the side of protein binding and insertion. The electrical field across the cytoplasmic membrane (in vivo) created by the electrochemical proton gradient favors poreformation. The structure of the open channel state is not known. The channel is formed by a single colicin molecule [reviewed in [19]]. In the open state of the channel, a significant part of the C-domain (68 residues, helices II-V of colicin la) becomes accessible to ligand binding from the trans side of membrane [16, 33]. The dependence of channel-formation by colicins on the lipid content of membrane points to the involvement of lipids in the formation and structure of ion channel that has been characterized as a "toroidal pore" [42]. Cytotoxic activity of endonuclease colicins also resides in the C-terminal domain. The size of the Cterminal cytotoxic domain of these colicins ranges from 96 to 135 residues, respectively. The immunity proteins bind these domains with very high affinit)^ (Kd < 10"^''M; [39]) efficiently blocking the interaction of colicins with their substrates, DNA or RNA. Extensive, highly complementary interaction surfaces in endonucleaseImm complexes that involve hydrophobic and polar side chains of both proteins, account for the highaffinity nature of Imm binding. Active sites in both DNase and rRNase colicins involve residues C terminal to the residues involved in Imm binding. This can allow Imm binding to an incompletely translated colicin in order to afford protection to host cells during the toxin synthesis.
MECHANISM OF IMPORT ACROSS OUTER MEMBRANE Colicins parasitize receptors that have a metabolic function in the binding and transport of vitamins, metals, and sugars. The best characterized colicin receptor system is that of vitamin B12 receptor (BtuB) utilized by colicin E3 (Table 1). The colicin binds tightly (Ka < 10"^^ M) to BtuB. A 2.75 A structure of BtuB-R-domain has been obtained [18]. Translocation of colicin E3
across the outer membrane appears to require a second outer membrane protein, the OmpF porin [2], to which the T-domain can bind, forming a two-receptor translocon for colicin import across the outer membrane. The coupled and concomitant events required for this import are (i) high-affinity binding to BtuB [37], (ii) unfolding of the colicin and extracellular release of immunity protein, (iii) insertion of disordered Nterminal region of the T-domain into the OmpF channel [41], and (iv) presumed cleavage of C-domain at an exposed R-C junction [9, 32].
AREAS OF RESEARCH ENCOMPASSED BY COLICIN STUDIES As just described, colicin studies span a range of general interests that include mechanisms of protein secretion, protein import, protein-receptor interactions, high-affinity protein-protein interactions, ion channel structures and function, and intracellular nuclease activities. Acknowledgment Studies of the authors reported here were supported by a grant (GM-18457) from the NIH.
References [1] Almendinger R, Hager LP. Studies on the mechanism of action of colicin E2, 107-33. In "Chemistry and function of colicins" (Hager LP, ed.). Academic Press, New York. [2] Benedetti H, Frenette M, Baty D, Lloubles R, Geli V, Lazdunski C. Comparison of the uptake systems for the entry of various BtuB group colicins into Escherichia coll J Gen Microbiol 1989;135:3413-20. [3] Bishop LJ, Bjes ES, Davidson VL, Cramer WA. Localization of the immunity protein-reactive domain in unmodified and chemically modified COOH-terminal peptides of colicin El. J Bacteriol 1985;164:237-44. [4] Bouveret E, Rigal A, Lazdunski C, Benedetti H. The N-terminal domain of colicin E3 interacts with TolB which is involved in the colicin translocation step. Mol Microbiol 1997;23:909-20. [5] Bowman CM, Sidikaro J, Nomura M. Specific inactivation of ribosomes by colicin E3 in vitro and mechanism of immunity in colicinogenic cells. Nat New Biol 1971;234:133-7. [6] Braun V, Patzer SI, Hantke K. Ton-dependent colicins and microcins: modular design and evolution. Biochimie 2002;84: 365-80. [7] CardelliJ, KoniskyJ. Isolation and characterization of an Escherichia coli mutant tolerant to colicins la and lb. J Bacteriol 1974;119:379-85. [8] Cavard D, Oudega B. (1992). General introduction to the secretion of bacteriocins, 297-305. In "Bacteriocins, Microcins, and Lantibiotics" (James R, Lazdunski C, Pattus F, eds.). SpringerVerlag, Berlin. [9] de Zamaroczy M, Mora L, Lecuyer A, Geli V, Buckingham RH. Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB. Mol Cell 2001;8:159-68.
CoLiciNs: BACTERIAL/ANTIBIOTIC PEPTIDES [10] Elkins P, Bunker A, Cramer WA, Stauffacher CV. A mechanism for protein insertion into the membranes is suggested by the crystal structure of the channel-forming domain of colicin El. Structure 1997;5:443-58. [11] Geli V, Baty D, Pattus F, Lazdunski C. Topology and function of the integral membrane protein conferring immunity to colicin A. Mol Microbiol 1989;3:679-87. [12] Gould JM, Cramer WA. Studies on the depolarization of the Escherichia coli cell membrane by colicin El. J Biol Chem 1977;252:5491-97. [13] Graille M, Mora L, Buckingham RH, van Tilbeurgh H, de Zamaroczy M. Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein. EMBO J 2004;23: 1474-82. [14] Hilsenbeck JL, Park H, Chen G, Youn B, Postle K, Rang C. Crystal structure of the cytotoxic bacterial protein colicin B at 2.5 A resolution. Mol Microbiol 2004;51:711-20. [15] Jakes R, Zinder ND. Highly purified colicin E3 contains immunity protein. Proc Natl Acad Sci USA 1974;71:3380-84. [16] Rienker PR, Jakes R, Blaustein RO, Miller C, Finkelstein A. Sizing the protein translocation pathway of colicin la channels. J Gen Physiol 2003;122:161-76. [17] Riihlmann UC, Pommer AJ, Moore GR, James R, Rleanthous C. Specificity in protein-protein interactions: the structural basis for dual recognition in endonuclease colicin-immunity protein complexes. J Mol Bio 2000;301:1163-78. [18] Rurisu G, Zakharov SD, Zhalnina MV, Bano S, Eroukova VY, Rokitskaya TI, Antonenko YN, Wiener MC, Cramer WA. The structure of BtuB with bound colicin E3 R-domain implies a translocon. Nat Struct Biol 2003;10:948-54. [19] LakeyJH, Slatin SL. Pore-forming colicins and their relatives. Curr Top Microbiol Immunol 2001;257:131-61. [20] Lazzaroni J-C, Dubuisson J-F, Vianney A. The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins. Biochimie 2002;84:391-97. [21] Lindeberg M, Zakharov SD, Cramer WA. Unfolding pathway of the colicin El channel protein on a membrane surface. J Mol Biol 2000;295:679-92. [22] Luria SE. On the mechanisms of action of colicins. Annales de L'Institut Pasteur 1964;107:67-73. [23] Nomura M. Mechanism of action of colicins. Proc Natl Acad Sci USA 1964;52:1514-21. [24] Ohno-Iwashita Y, Imahori R. Assignment of the functional loci in colicin E2 and E3 molecules by the characterization of their proteolytic fragments. Biochemistry 1980;19:652-59. [25] Parker MW, Postma JPM, Pattus F, Tucker AD, Tsernoglou D. Refined structure of the pore-forming domain of colicin A at 2.4 A resolution. J Mol Biol 1992;224:639-57. [26] Phillips SR, Cramer WA. Properties of the fluorescence probe response associated with the transmission mechanism of colicin El. Biochemistry 1973;12:1170-76. [27] Postle R, Radner RJ. Touch and go: tying TonB to transport. Mol Microbiol 2003;49:869-82. [28] Reeves P, ed. (1972). "The Bacteriocins," Chapter 1. SpringerVerlag, New York.
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