The membrane channel-forming colicin A: synthesis, secretion, structure, action and immunity

Biochimica et Biophysicn Acta, 947 (1988) 445-464

445

Elsevier BBA 85336

The membrane channel-forming colicin A: synthesis, secretion, structure, action and immunity Claude J. Lazdunski a, Daniel Baty a, Vincent Geli a, Danielle Cavard a, Juliette Morion a, Roland Lloubes a, S. Peter Howard a, Martine Knibiehler a, Martine Chartier a, Stanislas Varenne a, Michel Frenette a, Jean-Louis Dasseux b and Franc Pattns b ° Centre d e Biochimie et d e Biologic Moldculaire d u C . N . R . S . . Marseille (France) a n d b E u r o p e a n M o l e c u l a r Biology Laboratory, Heidelberg (F.R.(3.)

(Received 22 December 1987)

Contents I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I1.

Synthesis of colicin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The C o l a plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Organization and transcription regulation of the genes for colicin production, release and immunity . . . . . . . . . . . . C. Translation of the messenger RNA for coficin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Apparent difference of conformation between cytoplasrnk~ and extracellular polypeptide chain of colicin A . . . . . . .

445 446

446 447 448

448

111. Extracellular release of coficin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

449

IV.

Structure of coficin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Domain organization and primary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Shape of colicin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Secondary structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

452 452 454

V.

lmmanogenicity and antigenicity of cnficin A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

454

Vi.

Evidence for a pH dependent interaction between domains and unmasking of lipid binding sites . . . . . . . . . . . . . . . . . .

455

454

VII. Colicin receptors and entry into sensitive cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

456

VIIi. Mechanism of action of colicin A and other channel-forming colicins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Channel properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Modelling of the channel structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Molecularity of the colicin channel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

457 457 460

IX.

Colicin immunity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

461

X.

Summary

462

..............................................................................

Correspondence: C J, Lazdunski, Centre de Biochimie et de Biologic Mol6culaire du C.N.R.S., 31 Chemin Joseph Aiguier, B.P. 71. 13402 Marseille Ced¢x 9, France. 0304-4157/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

446 Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

462

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

462

!. Introduction Colicins are toxic proteins produced by and active against Escherichia cod and closely related bacteria. They are produced in large amounts and, in general, are secreted across the cell envelope to the extracellular medium. They can adsorb to specifi~ ~e~ptors i,~¢ated a~ the external surface of the outer membrane of sensitive cells, and are then translocated to their specific targets within these cells. Colieins were discovered a long time ago and were studied intensively until the sixties [1]. After a period of relative neglect, they have now emerged as powerful model systems for investigation of the mechanism and the energeties of protein insertion into and transfer across membranes. Colicins are encoded by plasmids that confer upon their hosts the property of being insensitive (one says immune) to the colicin produced. Immunity proteins are generally of low molecular weight (less than 20000). The largest group of colicins comprises those which can form voltagedependent channels in membranes, thereby destroying the cell's energy potential. This group includes colicins A, B, El, Ia, Ib, K and N. Immunity to pore-forming colicins may either prevent the colicin from spanning the cytoplasmic membrane, or prevent the pore from opening [2]. Much information has been obtained in recent years on colicin A and its immunity protein and it is of interest now to relate all this information dealing with synthesis, secretion, structure, antigenicity, mode of action, immunity, etc. in an integrated picture. For purposes of comparison, relevant data on other baeterioeins will also be discussed. However, since the various aspects cited above for bacteriocins have been previously the subject of reviews dealing specifically with one point or another, the reader is referred to these for wider aspects of colicinogeny [3-5]. What is the interest in studying cohcinogenie plasmids and colicins? It turns out that some interesting concepts of modem molecular biology are derived from such studies. The colicinogenic

plasmids by themselves have allc,wed us to elucidate processes such as the control of D N A replication [6] and plasmid copy number [7], plasmid incompatibility [8] and stability [9], etc. With regard to the synthesis of colieins, the demonstration of the fact that translation is a non-uniform p r o ~ s s related to codon usage was also ba.~ed upon studies dealing with colicins [10,11]. A new system for protein secretion has been discovered by studying colicin synthesis [12,13]. Two fields will benefit from colicin studies in the near future. The energetics of protein transfer across and insertion into membranes and the deetrophysiology of voltage-sensitive ion channels I14,151. II. Synthesis of coliein A II-A. The ColA p la smid

The complete nucleotide sequence of the multicopy C o l a plasmid has been determined [16]. This

xo~

~P-ma re . - ~".~-~.~] . . . .

~/"oe,,oo

. . . . N ~ /

S

W

~

\

'

k

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////7 I Fig. 1. Map of Cola and sites of insertion o! transposonst ' ) (Tnl or Tn3). The previouslydetermined [18] orientations of transcription of cart, cai and cal are indicated by bold lines and arrows

447 plasmid consists of 6720 bp. A t this m o m e n t 15 biological functions have been identified a n d are s h o w n o n the functional m a p (Fig. 1). These ind u d e 11 genes a n d three specific D N A sites. N i n e genes encode proteins of which three have been fully characterized. T w o genes encode u n t r a n s lated R N A s which are involved in the initiation of plasmid replication a n d copy n u m b e r control. T h e regions involved in the control of replication, plasmid mobility a n d stability have been identified using t r a n s p o s o n insertion mutagenesis a n d c o m p l e m e n t a t i o n [17]. This resdew will deal with three gene products from pCoL~: the gene for c~l.i~n A (caa for colicin A activity), the g~nc for ~olJ~itl A inb-nunizy (cai) a n d the gene product required for eolicin release (cal for colicin A lysis). H-B. Organization and transcription regulation of the genes for colicin production, release and i.,nmuni 9,

T h e initiation sites a n d terminators of transcription for the three genes caa~ cai a n d cal have been analyzed b y ~mclease S1 m a p p i n g . T h i s anal-

ysis demonstrated, that c~,2 a n d cal form an operon; cai in located between these two genes atz.d transcribed in the opposite direction from its o w n promoter. LexA protein, the repressor of chrom o s o m a l SOS-regulated genes [18], strongly repressed the in vivo a n d i.n vitro transcription of the caa-cal operon. A s determined b y D N A a s e 1 protection experiments, L e x A protein b i n d s with a high affinity to a n approx. 40 b p long sequence j u s t d o w n s t r e a m the Prib,aow box. T h e sequence of the b i n d i n g site is c o m p o s e d of two overlapped ' S O S boxes'. T w o transcripts of the caa-cal operon were detected by blot hybridization. T h e 19~o,¢r ~a~.R..~A~c~_,-a_dlr~c? tbe ~ynthes~s c,f bokh co~cin A a n d the lysi~ protein while the shorter o n e is terminated at t h e e n d of caa. W h e n the transcription of the caa-cal operon is induced, there is a s t r o n g interference with cai transcription (Fig. 2). T h e s a m e t e r m i n a t o r D N A region is used for the caa and cai genes. I n the caa direction, this bidirectional terminator h a s the characteristic structure c f a r h o - i n d e p e n d e n t terminator, it arrests a b o u t 50% of t h e transcripts initiated. T h e t e m | i , a t o r region for die ,:aa-cal transcript h a s a

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~

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~

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Fig, 2. C~t~,~dL.Q;,~v, of the caa-cal operon and the cai gene. The nucleotide sequences for the promoters are presented: the

recognition and binding sequences for RNA polymerase are boxed with dotted lines (-35 sequence) and su!id lines (Pribunw box). PA and SA indicate the Priboow box and transcriptional start site for the can.col operor, while PI and Si ind;cate those for the cai gene. SI = indicates the start site for cai in vitro. Arrows above the start sites indicate the direction of transcription, the bold line indicates the major band detected by Sl mapping. In the case of S1*, one of the uncleotides within the bracket corresponds to the start site. The binding site for LexA in the operator region for can is indicated by brackets overlining file protected region for the sense-strand and brackets underlining the sequence (not represented) for the anti-sense strand. The two overlapped consensus 'SOS sequences"are underlined. The sequence is numbered taking the cleavage site for Hincll as the + l nucleotide. Coding sequences for can, ca# and cal are indicated by dotted and hatched boxes. Numbers above the boxes indicate the first and last nucleotides of the coding sequence, and arrows indicate the direction of transcription. Transcripts of caa and coa-cal are indicated as well as the terminators T1 and T2 (T3 is identical to T1, but located on the opposite strand).

448 rate of chain elongation during colicin A biosynthesis was not uniform. This feature also applied to colicins E2 and E3 [10]. A detailed analysis provided evidence that this p h e n o m e n o n is not related to aspects of messenger R N A such as secondary structure. It is linked to the difference in transfer R N A availability for the various codons. Experimental analysis indicated that this is a general feature for the translation of all m R N A s in E. coil The degree of slackening in ribosome movement is almost proportional to the inverse o f t R N A concentrations. It so happens that colicin genes contain a high proportion of codons corresponding to rare t R N A s and this explains why intermediate nascent chains could be easily observed [11] (Fig. 3).

complex structure and may contain elements for both rho-~ndependent and rho-dependent termination thereby insuring efficient separation of transcription of the c.aa-cal D N A region from the region involved in the control of D N A replication (Lloub6s, R., Baty, D. and Lazdunski, C., unpublished results).

II-C. Translation of the messenger RNA for colicin A Colicin A is synthesized in free polysomes in the cytoplasm [19] and although it is a secreted protein it is not produced in a precursor form [19,20]. It is produced in very large amounts after induction and its synthesis accounts for more than 50% of total protein synthesis, probably because the caa promoter is very efficient and more than 20 copies of the gene are contained per cell [16]. Early in our studies on colicin A production, we observed that discrete intermediates in polypeptide elongation appeared during synthesis [19]. Extension o f these studies demonstrated that the

1

2

3

4

5

II-D. Apparent difference of conformation between cytoplasmic and extracellular polypeptide chain Qf colicin A By using pulse labelling and chase, we have shown that as long as less than 450 amino acids o f

6

m

_a am

Fig. 3. Elongation intermediates of colicin A polypeptide. Samples from 10-s pulse-labelled cells (lane 1) were removed at different times of chase (lanes 2-11). Samples were solubilized, immunoprecipitated and analyzed on a SDS-polyacrylamidegel: lane 2,10 s; lane 3, 20 s; lane 4, 30 s; lane 5, 40 s; lane 6, 50 s; lane 7, 60 s; lane 8, 70 s; lane 9, 80 s; lane 11,100 s. The migrations of colicin A monomer and dimer are indicated by arrowheads.

449 the 592 residues of colicin A polypeptide chain have been assembled, the nascent chains are soluble and have no affinity for membranes. Beyond 450 residues, the nascent chain acquires a COOH-terminal region, comprising a 48 amino acid hydrophobic stretch, which is involved in pore formation [21]. At this point, the nascent chains acquire two new properties. Firstly, they become competent for membrane insertion [11] and secol,dly, they can form stable dimers of colicin A [22]. The nascent chain intermediates resulting from pause sites and the dimer form are shown in Fig. 3. As soon as they are secreted to the extracelhilar medium, the high dilution from 0.7 .am3 (intracellular volume) to the 'pacific ocean' (the extracellular medium) appears to promote a slow conformational change by which the polypeptide loses its competence for membrane insertion and the ability to form dimers. In this process, the COOH-terminal hydropimbic stretch most probably becomes masked in the interior of the protein. To explain our results, we were led ~,, h)~pnthesize that the same polypeptide region which confers competence for membrane binding on the cytoplasmic colicin A (see below) also confers the ability to form dimers. These dimers are remarkably stable, since they are not dissociated in the presence of high concentrations of SDS as long as the protein is not heated [22]. HI. Extracellular release of colicin A

Using transposon insertion mutagenesis or via deletion of the lysis gene, strains deficient in the production of Cal protein (51 amino acid residues) can be obtained and these cells do not release colicin A, thus demonstrating the role of this protein in colicin release. We have observed that a critical concentration of Cal is required to trigger colicin release and, deletion of the T1 terminator causes overproduction of Cal, and an early decrease in the turbidity of the culture with concomitant loss of viability. Although cells which are excreting colicin do not show signs of extensive disintegration [20,23], they do exhibit a number of features which indicate that envelope functions are affected. They are no longer able to accumulate labelled substrates,

they release ions from the cytoplasm and then are sensitive to the osmotic strength of the medium, which suggests that the inner membrane has become permeable to small m~lecules [24]. These effects can be ~lmost completely overcome by adding 20 mM Mg 2+ to the medium. This treatment also prevents the decline in culture turbidity, but was reported to have no effect on colicin export [24,25]. Some colicins are released as a complex with their immunity protein. However, it has been demonstrated that the cloacin DF13 immunity protein was also produced and excreted in the absence of cloacin molecules [25]. The structures of the known bacterial release proteins feature a high degree of homology (Fig. 4 ) The se0uenees of the lysis proteins encoded by eight different Col plasmids reported so far have been compared. The characteristic features are an N-terminal signal peptide with a sequence, at the cleavage site, resembling the consensus lipoprotein modification sequence described by Wu and Tokunaga [31], followed by a highly conserved stretch of 19 amino acids. Studies on the ColE2 and CclF3 l~/sis proteins [26,32] as well as recent data on the CoIN lysis protein [33] suggest that their C-terminal tails are not m~oortant for their biological activity. This is also suggested by t~he fact that the C-terminal regions of the CoIN and ColA lysis proteins are different from those of most other lysis proteins (Fig. 4). Although a conserved 20 amino acid region appears to exist, the underlying structural requiremeats for activity are not yet clear, because sitedirected mutaganesis studies indicate that a number of amino acid residues can be substituted without significant loss of activity (S. Howard, D. Cavard and C. Lazdunski, unpublished data). For Cal, we have demonstrated that the precursor must be modified by a lipid before it can be processed and that the maturation is prevented by globomycin, an inhibitor of signal peptidase II [34]. Using ofigonucleotide-directed mutagenesis, the alanine (mutant AK31) and cysteine (mutant ALl6) residues in the -1 and + 1 positions of the cleavage site were replaced by proline and threonine residues, respectively, in two different constructs (Fig. 4). Both substitutions prevented the normal modification and cleavage of the parent protein (Fig. 5).

450 CONSRR~'ZD A

I~I~ON

MKKIIICVILLAIMLLAA~CQVNNVRDTGGGSVSPflSIV

DFI3

TGVSMGSEGVGNP

MKKAKAIFLFILIVSGFLLVATCQANYIRDVQGGTVAPSSSS

ELTGIAVQ

E1

MRKKFFVGIFAINLLVGTCQANYIRDVQGGTIAPSSSS

KLTGIAVQ

E2

MKKZTGIILLLLAVZ~LsATcQANYZ~DVQGGTVSPSSTA

EVTGLATQ

E5

MKKITGIILLLLAVIZLSA~CQANYZRDVQGGTVSPSSTA

EVTGLATQ

E5

MKKITGIILLLLAAIILAATCQANYIRDVQGGTVSPSSSA

ELTGLATQ

E8

MKKITGIILLLLAVIILAATCQANYIRDVQGGCVSPSSTA

EVTGLATQ

N MCGKILLILFFIMTLSATCQVNHARDVKGGTVAPSSSS RLTGLKLSKRSKDPL Fig. 4. Comparisonof the ~minoacid sequencesof the precursorsof Col plasmid lysis proteins. The sequencesare aligned with respectto the cysteineresidues(+ 1) whichformthe N-terminusof the matureprotein.Data are taken fromRefs.23, 26-30.

Although the detailed mechanism of release of colicins is not yet known, the increase in envelope permeability observed late after mytomycin C induction may be attributed, in part or totally, to the lysis protein-dependent activation of the detergent-resistant phospholipase A present in the outer membrane [24,35].

The marked activation of the detergent-resistant phospholipase A observed with wild-type Cat was not observed with the Cal mutants AK31 and ALl6. Both Cal mutants were also defective for the secretion of coficin A (Fig. 5) [34]. The i n c r e a s e in l y s o p h o s p h a t i d y l e t h a n o l a m i n e (lysoPE), the product of the action of phosphoriC 4011

A

AIHrll

AILlO

~151B

o

D-

O

2 4 Time(h)

6

8

O 1 2 3 4 5 T i m e ( h afteP induction)

4:10 ll~ P

$

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4 ~ II10 I

P

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Fig. 5. Effect of mitomycin C induction on growth end lysoPE content of cells containing wild-type and mutant Cat protehls. The growth cube (A) and the amount of lysoPE as a proportion of total phospholipids (B~ are presented. The cells were grown to absorbance at 600 nm of approx. 1 and were either not induced or induced with ndtomycin C. The phosphollpids were extracted and analyzed after further incubation. The plesmids and the induction conditions of the cells are as follows: X, W3110 (pAEII) wild-type induced; e, W3110 (pAEll) non-induced; +, W3110 (pAK3I) induced; o, W3110 (pAL16) induced. (C) The modification and processing of wild-type and mutant Ca] proteins, as well as the release of collcin A were analyzed. The cells were induced with mytomycin C for 90 rain before labc~ng with [35SJmethloldne. Then, at various times (as indicated), culture samples were centrifuged, and the pellets (P) and supematants (S) were analyzed by SDS-polyacrylamide gel electrophoresis. A fluorogram is presented. The various Ca] protein derivatives are indicated: O, Cal modified precursor form; o, Cal unmodified precursor form; Ip, Ca] mature form; I~, Ca] signal peptide. The position of colicin A and ~-Iactamas¢ are indicated.

iT6 fd'IINCOI Pill

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~

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.

~71 172 t73 ~Usl) (GIv) Gtu ATG.GJGG.GAA

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Fig. 6. Construction of recombinant plasmids. A part of the plasmid pColA9 is shown, as well as the constructions derived from it.

The restnction sites are indicated above the number of the cleavagesite. The oligonucleotidesused for the constructionsare boxed and the .,estrictionsi,~ ¢re~'.c~arc underlined.Stars represent stop codo~ S.D. is the Shmc-Dalg,~ansequence.The amino acids in brackets were created during the construction. AY? represents two protons AY,"N and AY7C. BF3 corresponds to the three mutationsintroducedinto AYTC[36].AY7Acontains the first 31 amino acids of A~'/N (calledAY?AN)and the AYTCprotein.

pase A on phosphatidylethanolamine, the major membrane phospholipid, was not observed (Fig. 5). LysoPE may, thus, act as a membrane perturbant which could alter the permeability properties of the envelope and allow colicin to leak out of the cell. In addition, a subset of at least 20 proteins is also released to the extracellular medium. Electron microscope studies with iramunogold labelling of coficin A on cryosections of pldA and cal mutants indicated that the coficin remains in the cytoplasm and is not transferred to the periplasmic space [34]. These results demonstrated that Cal must be modified and processed to activate the detergent-resistant phospholipase A and to promote release of coficin A. However, so far, the mechanism of transfer of coficins across the inner membrane remains poorly understood. In partiealar, it is not known if any interaction between a given colicin and its lysis protein is involved, or if any specific regions or specific interactions between regions of *.he colicin polyo peptide chain are required for this transfer. We

have now addressed this question by constructing deletion mutants of colicin A and by a new approach using a cassette which contains a stop codon, a Shine-Dalgarno sequence and an initiation codun to separate the NH2- and COOHterminal regions of colicin A. Together, these deletions span the region from amino a~id 15 tu the end of the protein (Fig. 6). None of these regions was found to be required for extracellular release or had any effect on the efficiency of this process. As well, the NH2-terminal and central plus COOH-terminal domains could be demonstrated to be released to the same extent when produced as separate polypeptides as when produced as linked ones. The introduction into the COOH-terminal domain of mutations promoting cytoplasmic aggregation [36] prevented its release but had no effect on the secretion of the" NH2-terminal polypeptide [37]. These results demonstrated that no specific interaction between the N H 2- and the COOH-terminal regions of the colicin A polypeptide chain is involved in the release

452 of coliciffA. We are led to conclude that there is no topogenic export signal in the polypeptide chain of colicin A involved in the release mechanism. Thus, the process is non-specific with respect to the colicin itself and depends solely on the expres. sion of the colicin A lysis protein. IV. Structure o~r eoliein A

IV-A. Domain organization and primary structure

It has been demonstrated that coiicins kill sensitive 17,. coil cells in three steps: (1) binding to a specific receptor located in the outer membrane; (2) trmasiocation across the membrane; (3) interaction with their specific target in the cell. The domains associated with the steps defined above are organized on a single polypeptide chain. Our studies have demonstrated that coficin A follows this general scheme. Various approaches have been used. We constructed hybrid proteins such as colicin A-fl-lactaraase [38] or deletion derivatives of colicin A, lacking various regions of the polypeptide chain (Fig. 6). Limited digestion of colicin A by proteinases, like bromelain and thermolysm, was also carded out (Fig. 7). The results of these studies indicated that the amino-terminal region is probably involved in translocation across the outer membrane. This can be inferred from the fact that derivatives lacking this region can bind to the receptor for colicin A and contain the pore-forming domain, but are nevertheless unable to kill sensitive cells. Trlnslocation

This N-terminal domain comprises at least 170 residues. It has now been purified and extensively studied (M. Knibiehler, V. Geli, D. Baty and C. Lazdunski, unpublished result). A short deletion of 15 residues (protein BD2) can suppress the in vivo activity. However, BD 2 can kill osmotically shocked cells, confirming that the translocation step is altered by the deletion. The receptor binding domain is located between residues 170 and 335 [38], and the pore-forming domain is comprised in the C-terminal domain from residue 386 to 592. This latter has been extensively studied. It can be prepared and purified in large amounts from colicin A by limited proteolytic digestion [21]. This domain is particularly interesting since it can exist in two different forms, a soluble one and a membrane inserted one. The primary structure of colicin A has been deduced from the nuclcotide sequence of the caa gene [39]. The colicin A polypeptide chain comprises 592 amino acids and has a molecular weight of 62989. The amino-terminal region is rich in proline and glycine residues and accordingly secondary structure prediction indicates that this region (1-185) is B-structured. The rest of the molecule [186-592] is predicted to be rich in a-helix. An uncharged amino acid sequence of 48 residues is located in the C-terminal part of the molecule [39]. In agreement with the domain organisation described above, colicin A shares homology with other colicins which can form voltage-dependent

Receptor Binding

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Function

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Co|icin A I BD2 ARI BE!

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70

3~o 389

Fig. 7. Functional organization of cofiein A polypeptide chain as defined by the properties of r~.ombinant )rotein constructs and proteolytic fragment. RB means receptor binding, the activity in vitro has been assayed in planar fipid bilayers [70].

453 A.Pept B.Pept N.Pept CONS1 Ei.Pept iA.Fept IB.Pept CONS2 Tot CONS C O N S (-i) C O N S (-2)

A.Pept B.Pept N.gept CONS1 EI.Pept IA.Pept IB.Pept CONS2 Tot CONS C O N S (-i) C O N S (-2)

1 50 VAEKAKDERE LLEKTSELIA GMGDKIGEHL GDKY~AIAKD IADNIKNFQG KKEQENDEKT VLTKTSEVII oVGDKVGEYLGDKYK~LSRE IAENINNFQG KEEKEKNEKE ALLKASELVS GMGDKLGEYL GVKYKNVAKEVANDIKNFHG --E .... E+- -L-K-SE ..... GDK-GE-L G-KYK---+ -A--X-NF-G KKAQNNLLNS QIKDAVDATV SFYQTLTEKY GEKYSI~AQ~ LADKSK*~*G EEKRKQDELK ATKDAINFTT EFLKSVSEKY GAKAEQLARE MAGQAK***G EEKRKRDEIN MVKDAIKLTS DFYRTIYDEF GKQASELAKE LASVSQ***G ............ K D A - - - T - - F . . . . . ._. G ...... A-E -A .... ***G ............................. ....... E ................... ---+--DE ...... A-_ ..........

G ......... 7--~ G - K . . . . A + E E~-- G - K Y - - - A + E

51 KTIRSFDDAM ASLHKITANP AMKINKADRD KTIRSYDDAM SS~KLMAN~ SLKiNATDKE R N I R S Y N E A M A S ~ ~MKVNKSDKD +-IRS-- AM -S-NK--ANP --K-N--D+_ KKIGN~AAFEKYKDVLNKKFSKADRD KKIRNVEEAL KTYEKYRADX NKKINAKDRA KQIKS%~)DAL NAFDKFRNNL NKKYNIQDRM K-I--V-AL ---_K-+--- NKK .... DR-

A .... ***G -A .... *~*G - A ....K ~ * G

I00 ALVNAWKHVD AQDMANK*** Ai%~AWKAFNAEDMG~-~ ~ AIVNAWKQVNAKDMANK*** A-VNAWK--- A-DM-NK*** AIFNALASg~KYDDWAKHLDQ AIAAALESVK LSDISSNLNR AISKALEAIN QVHMAENFKL AI--AL ..............

+ . .I. . . _ A - . . . . K . . . . . . . . K . . . . D + - A - - - A . . . . . . . . . . . . *** K-I+--- A ..... K ....... K-N--D+- AI--A ....... D .... *** K-IRS---A ..... K--AN- --K-N--DRAI-NA---V--DMA--***

A.Pept B.Pept N.Pept CONS1 EI.Pept IA.Pept IB.Pept CONS2

i01 ****LGNLSK AFKVADVVMK VEKVREKSIE ****FAALGK TFKAADYAIK ANNIREKSIE ****IGNLGK AFKVADLAIK VEKIREKSIE ****---L-K -FK-AD---K .... REKSXE FAKYLKITGH VSFGYD%"~SD ILKIKD**** FSRGLGYAGK FTSLADWI~Z FC:.~VR**** FSKAFGFTGK VIERYDVAVE LQKAVK**** F - + . . . . . G . . . . . . D . . . ._ . K ---e***

150 GYETGNWGPLMLEVESWVLS GYQTGNWGPL MLEVESWVIS GYNTGNWGPL LLEVESWIIG GY-TGNWGPL -LEVESW--***TGDWKPL FLTLEKKAAD ***TENWRPL FVKTETIIAG ~**TDNWRPF FVKLESLRAG "**T--W+P- F---E---A-

Tot CONS C O N S (-1) C O N S (-2)

**** ........... D .......... **** **** .... GK ..... D ...... K---**** ****-G--GK .... AD ...... K-+ _ *~**

***T--W-P ..... E ..... ***T-NW-PL .... E ..... ***TGNW-PL -L--ES ....

A.Pept B.Pept N.Pept CONS1 E1.Pept IA.Pept IB.Pept CONS2

151 GIASSVALGI GMASAVALSL GVVAGVAISL G .... VA--AGVSYVVALL NAATALVALV RAASAVTAWA ....... A--

200 FSATLGAYAL SLGVPAIAVG IAGI*LLRAV VGALIDDKFA FSLTLGSALI AFGLSATVVG FVGV*VIAGA IGAFIDDKFV FGAVLSFLPI S*GLAVTALG VIGI*MTISY LSSFIDANRV F---L ....... G ...... G --G-* ......... ID .... FSLLAG**** ******TTLG IWGIAIVTGI LCSYIDKNKL FSILTG**** ~**~**SALG IIGYGLLMAV TGALIDESLV FSVMLG**** ******TPVG ILGFAIIMAA VSAL~/NDKFI FS---G**** ******---G I-G .................

Tot CONS C O N S (-I) C O N S (-2)

.......... ..... V .... --AS-V ....

F ..... **** FS---G**** FS--LG****

A.Pept B.Pept N.Pept CONS1 EI.Pept IA.Pept IB.Pept CONS2 Tot CONS

201 212 DALNNEIIRP AH DELNHKIIK. .. SNINNIISSV IH ---N--I ..... NTINEVLGI. .. EKANKFWGI. .. EQVNKLIGI. .. ---N---GI ---N .....

******---G ******---G ******T--G

--G-* ............... --G-* ......... ID .... I-G-* ....... A-ID_---

Fig. 8. Comparison of the amino acid sequences of the COOH-terminal domains of channel-forming coficins. Data are taken from Refs. 32, 39, 91-93. Pore-forming colicins have been divided into two subgroups, group 1 comprising A, B, N and group 2 comprising El, 1^ and I m The consensus sequences for each group and for the two groups are indicated. Stars indicate deletions and dashes in the conseusus sequences indicate lack of homology.

454

channels in membranes only in the C-terminal end of the polypeptide chain. This group includes colicins A, B, El, Ia, Ib, K and N. Within this group sequence comparisons suggest the existence of two sub-groups (Fig. 8). Colicins A, B and N share an extensive homology while coHcins El, Ia and Ib seem to belong to a second subgroup. There is experimental evidence that colicins A, N, B, E1 and la can be also classified into the same two groups according to their channel properties [40-44!. However, all those colicins have a number of features in common. First, they all contain a 35-48 amino acid region containing only uncharged or hydrophobic amino acids near tl'..~ C-terminus. Second, the early region of the C-terminal domain (see below) is always rich in lysine residues with a strong bias against arginine. High structural homology is also suggested by the hydrophobicity profiles and the correspon,-ting correlation coefficients (Ref. 45 and F. Pattus, unpublished results).

IV-B. Shape of colicin A The hydrodynamic properties of coficin A have been studied in detail [46]. The molecular weight determined from sedimentation equilibrium centrifugation was found to be 63000, in agreement with that determined from the primary amino acid sequence [39]. The sedimentation coefficient has been analyzed over a wide range of ionic strength (NaCI 0.06-0.56 M) and pH (8-4) and was found to remain almost constant. However, below pH 4, a colicin A tetramer is spontaneously formed. This micelle-like protein aggregate probably arises after unmasking of putative hydrophobic and amphipathic a-helices [45]. The tetramer can be dissociated (into monomers) in the presence of an excess of lipid. These monom~,rs were shown to be associated to 22 lipid monomer mo|ecu!e~s0 suggesting that a h:Lgh-affinity lipidbinding site is involved in tetraraer assembly [46]. The frictional coefficient value indicated that the shape of the coficin A monomer was very elongated .:~i~ values of 8.1 for an elongated prolate or ellipsoid and 9.6 few a flat oblate or ellipsoid. For the tetramer, the values were 15.4 and 20.8, respectively. These axial ratios are similar to those calculated for other pore-Jorming col-

icins El, la, Ib and K [47]. These coficins appear to be more highly elongated molecules than those having nnclease activity, which are isolated in association with their immunity proteins [48]. However, colicin E3, which is a ribonuclease, forms a dimer after removal of its immunity protein [48] and the dimer has an axial ratio of 9-11, similar to that of the colicin A monomer. Such a correlation might be fortuitous, but it is tempting to sp~ulate that the highly eioagated shape may play some role in colicin function. In contrast, analytical ultracentrifugation of the C-terminal thermolysin peptide in solution showed that the pore domain is globular.

IV-C. Secondary structure Conformational investigations, using circular dichroism, have been carried out on colical A and on the C-terminal bromelain and thermolysin fragments (molecular weight 20000) to estimate their secondary structure and to search for pH-dependent conformational changes. Colicin A and the bromelain peptide are mainly a-helical with a n enrichment of the a-hefical content in the Cterminal domain carrying the channel-forming activity (see below). The non-negligible t-sheet structure in the C-terminal domain is unstable and is easily transformed into a-helix upon decreasing the polarity of the solvent. There is no drastic change in secondary structure in the presence of neutral detergent. Negatively charged phosphofipids promote the same changes as decreasing solvent polarity [45]. Secondary structure predictions have also been carded out [45]. The results will be discussed in the subsection VIII-B. V. lmmunogenicity and antigenicity of colicin A As an additional approach to understanding the function of coficin A, monoclonal antibodies directed against this protein were prepared. Several hundred hybridoma cell cultures were screened using an immunoadsorption procedure. The growth medium of 84 cultures gave a positive result. Among these, 15 cultures were selected and the corresponding cell lines were cloned and used to produce monoclonal antibodies [49].

455 Five out of the 15 monoclonal antibodies could preferentially recognize wild-type colicin A as compared to derivatives obt~ued by genetic engineering. These derivatives (see sabsection IVA) were used to map the epitopes of te~ monoclonal antibodies. It was striking to observe that most of the monoclonal antibodies were directed against C- and N-terminal regions that contained uid~ h,,~: of the predicted epitopes of the polypeptide eh ;ain. This might be finte~re:cd a~ meaning that the C- and N-terminal regions constitute surface domains protruding from the general surface structure. Some of the antibodies did not bind to colicin A when it was preincubated at acidic pH. These results are in perfect agreement with studies on the citculax dichroism and hydrodynarmc p:opc: ties of eolicin A [46]. All of these studies suggest that the colicin A polypeptide chain undergoes a conformational change below pH 4 resulting m exposure of a hydrophobic region and subsequent tetramerization of colicin A. The interactions of colicin A, its NH2-terminal, and its COOH-termihal domain with phospholipid monolayers at neutral and acid pH values suggested that an interaction between the polypeptide region 173-336 and a hydrophobic region from the COOH-terminal domain is disrupted at acid pH (see below). ¥1. Evidence for a pH dependent interaetion between domains and unmasking of lipid binding sites

The mechanics of colicin uptake are poorly understood. This is a difficult problem since the number of celicin molecules entering a cell is below the level of detection and a single colicin molecule is theoretically sufficient to kill a sensitive ceil. Since translocation through membranes occurs during uptake, membrane lipids may play a role in one or more of the steps described above. We have studied the interactions of colicin A Wltl~ ~p~ds m model membrane systems. Colicin A can insert into phospholipid monolayers, although at neutral pH the aff'mity is not high. In contrast, below pH 5.5, this affinity is considerably increased and colicin A becomes a highly penetrating protffm, as strong as the lyric peptides meilhin and cardiotoxin [501. This interaction seems to be governed by groups with a pK of 5.5. Similar

results were obtained with lipid vesicles [5i]. Moreover, colicin A was found to promote efficient fusion of lipid vesicles at acidic pH. Fusion was not only observed with pore-forming colicins (A, El) but also with colicins that contain nuclease activities (E2, E3). The appearance of a lipid-binding site in colicin E3 at acidic pH has been demonstrated by 'Escuyel et al. [52]. These results as well as those obtained with monocloaal amibodies (Section V~ and the existence of a pH-de.:9~dent equilibrium between two forms of file colicin A ionic channel (see Section VIII), may be interpreted by assuming the existence of a pH-dependem asso-,-iation between two domains in the colicin A molecule. The existence of a low-affinity lipid-binding site at neutral pH is also indicated by the differences in the digestien patte~ •f colicin A by proteinase V8 in the presence or absence of detergent [46]. As well, hydrodynamic studies indicated that the colicin A monomer at neutral pH can bind to lipid micelles [46]. Careful comparison of the ratios of the increase. in surface pressure of lipid films at neutral and acidic pH values indicated that the COOH-terminal domain (thermolysin fragment, Fig. 7) had the highest activity for insertion into phospholipid monolayers. In contrast, the NH2-terminal domain by itself (172 amino acid residues) had a low affinity for the lipid film and this affinity was not increased at acid pH. The protein AR1 (deletion from 32 to 172) behaved like the colicin A, while protein BE1 (deletion from 30 to 371 see Fig. 7) behaved like the thermolysin fragment (Frenette, M., Knibiehler, M.. Baty, D., Geli, V., Pattus, F., Verger, R. and Lazannski, C., unpublished results). The properties of the deletion mutants in monolayers, planar bilayers [40] and in the vesicle fusion assay (Frenette, M., Knibiehler, M., Baty, D., Geli, V., Pattus, F., Verger, R. and Lazdunski, C., unpublished results) suggest that there is an interaction between a polypeptide region, lc,cat-c,d between residues 173 and 336, with the COOHterminal domah~. Disruption of this interaction at acidic pH would unmask a new lipid-binding site on d~e central domain of the chain responsible for the fusion activity of the colicin and produce the 'acidic' form of the channel (see subsection VIII-

456 A). This new lipid-binding site which is unmasked at lower pH on colicin E1 and E3 than on colicin A may play a role in the translocation step through membranes. This hypothesis implies, however, the presence of an acidic compartment in E. coil within the periplasmic space. These experiments and the results from hydrodynamic studies described above suggest that there eu.c two locks in the molecule, one between the central and the COOH-terminal domains of colicin A and the other located within the COOHterminal domain itself. At pH 5, one lock would be disrupted, thus allowing exposure of a hydrophobic domain at the surface of colicin A and conferring increased affinity for lipid films. Below pH 4, the second lock would be disrupted (circular dic_hroism does indicate an unfolding) with unmasking of another lipid binding site. The latter is presumably involved in the formation of the tetramer and in the dissociation of tetramer to monomer in the presence of an excess of lipid [46]. All these studies should help to understand the stepwise pathway through which colicin A first interacts with its receptor at the cell surface, is then translocated across the outer membrane and finally reaches the cytoplasmic membrane to in~;ert its COOH-terminal domain. In this process, the colicin A polypeptide chain must switch from a water-soluble conformation to one that is more stable in the membrane. Our studies suggest that a partially exposed lipid-binding site may become more exposed at acid pH when the interaction between the NH2-terminal dcmain and the COOH-terminal domain is disrupted. Then a further acidification may cause further unfolding with exposure of a new high-affinity lipid-binding site. Cohcin A mutants altered in the COOH-terminal region have been found to spontaneously aggregate in the cytoplasm of producing cells [36]. This result also suggests that a rather discrete conformationai change might be needed to expose the high-affinity lipid-binding site of colicin A. The polypeptide chain of pore-forming colicins conrains a large hydrophobic stretch of 35-50 residues in the COOH-terminal region [53]. Putative membrane spanning hydrophobic helices of about 20 residues each have been predicted m this region for colicin E1 [53] and colicin A [45]. It is, thus, tempting to hypothesize that either the formation

of helical hairpins or their exposure is favored at acid pH. The spontaneous insertion of such a hydrophobic helical hairpin into the membrane would be energetically favored, as pointed out by Engelman and Steitz [54]. Vll. Colicin receptors and entry into sensitive cells As previously mentioned, once they ate released from producing cells, colicins can bind to specific receptors on *.he surface of susceptible cells [3]. These receptors are comprised of outer membrane proteins, called porins, that allow the diffusion of low-molecular-weight nutrients across the outer membrane. These porins have been parasitized by bacteriophages and colicins which use them as receptors. The colicin-receptor interaction is energy-independent and can occur below the transition temperature of the membrane. The receptors for pore-forming colicins are shown in Table I. The expression of most of these receptor proteins is regulated by growth conditions. Many of the colicin receptors have been shown to be involved in outer membrane-mediated nutrient uptake. Thus, the polypeptide that serves as the receptor for colicins E1 and A (BtuB) functions in the uptake of vitamin B-12, whereas the colicin K receptor serves as a specific diffusion pathway for nucleosides. Several colicin receptors are involved in iron uptake, serving as siderophore-binding proteins. These important physiological functions exert selective pressure for the maintenance of active receptors on the surface of sensitive organisms.

TABLE 1 RECEPTORS FOR PORE-FORMING COLICINS. Colicin Genetic Molecular Other hgands locus weight or nutrients A t~mBompF 60000 vit. B-12 37000 various< 700 B fepA (cbr) 8 1 0 0 0 enterochelin E1 btuB 60000 vit. B-12 la Ib cit 74000 iron accumulation K tsx 27900 nucleosides N ompF 37000 various< 700

Ref. 55 56 57 3 58 59

457 The molecular events between the initial adsorption to receptors and final interaction with a particular cellular target, is not known for any colicin. However, there are indications that such translocation may be energy-dependent. According to its activity spectrum against a variety of mutants, a particular colicin can be unambiguously assigned to one of two groups. The type 'B' colicins (B, Ia, Ib, V, D and M) are inactive against strains that have a lesion in the tonB gene, but active against strains mutant in tom or tolB genes, whereas the type 'A' colicins (A, El, K) show the opposite specificity. This distinction is independent of mode of action, but is thought to reflect two different modes of colicin uptake. Although the exact function of the tonB gene product has not been determined, available information has led to the consensus that it plays a role in mediating transfer of outer membranebound nutrients (such as vitamin B-12 and sidetophores) or type 'B' colicins from their respect;.ve surface receptors to the cytoplasmic membrane (for a review, see Ref. 3). The receptors can be bypassed since osmotic shock alleviates the need for receptors in cells challenged with colicins E3, M or E1 [60,61]. Under these conditions, the TonB function is no longer needed, suggesting its involvement in the translocation across the outer membrane. It is thought that the TonB protein is somehow involved in the energy-coupling process betw~.ce inner and outer membranes. The product of the gelie exbB is required fqr the vitamin B-12 and iron transport and also for the uptake of various colicins [56]. A homologous region ((Glu or Asp)-Thr-ValIle-Val) at the amino-terminal end of the mature form common to all TonB-ctependent receptor proteins has bern demonstrated [62]. The TonB protein was found in both the cytoplasmic and outer membrane and it has been proposed that this protein might form intermembrane bridges thus regulating the activity of outer membrane proteins according to the energy state of the cytoplasmic membrane [62,63]. For group 'A' colicins, the possible invoh,emer.t of new gene products for uptake in addition to TolA and TolB, has been suggested. Two open reading frames, tolQ and tolR proximal to the TolAB region have been described. The proteins

coded by this region comprise a system which allows transport of iarge molecules such as cob icins or filamentous phage DNA, into or across the bacterial membrane [64,65]. It has been proposed that colicins El, A, K and N as well as the gene IIl protein of fl phage, all of which feature a glycine-rich region at the amino terminus, may be interacting with the protein products of tolQRA gene chister. Ohno-Iwashita and Imahori [66] showed that the carboxy-tcrminal two-thirds of colicin E1 can kill osmotically shocked btuB cells but not intact btuB cells. They suggested that the amino-terminal portion of colicin E1 is requ;~red for its transport through the outer membrane after the receptor binding step. It is also of interest to point out that only sensitive cells with an energized membrane can be killed. This requirement applies not only to the formation and opening of channels for pore-forming colicins (see further) but also to coficin E3, which has its target in the cytoplasm [67]. Therefore, a potential across the inner membrane is probably also required for the transport of TonB-independent coficins across the outer membrane. Although the comparison m i # t be fallacious it is tempting to suggest that r :ns conform to the rules that apply to protein aansfer across membranes in mitochondria, the rough endoplamfic reticulum membrane and bacteria in general. The three steps, association with a receptor on the correct membrane, translocation, and folding on the opposite membrane surface also apply to colicins. The energy requirement for colicin uptake may be related to unfolding which could explain the lack of disulfide bridges in their polypeptide chains. This might hold true for both TonB-dependent and TonB-independent coficins. VHI. Mechanism of action of coliein A and other

channel-forming colicins VIII-A. Channel properties What is the structure responsible for the action of colicin A and other pore-forming colicins on the cytoplasmic membrane of sensitive cells? To investigate this point, planar lipid bilayers have been used [40,41-44,68-70]. With this system, known voltages can be applied across the

membrane and the polygepride insertion and the channel properties can be probed through conductance measurements. Colicins A, El, K, Ia and Ib can form voltage-dependent single cSarmels as demonstrated by the stepwise increase in conductanee observed after injeetion of very low concentrations of colicin (Fig. 9). These channels are functionally sim/!ar to channels from excitable membranes, although their ion selectivity is not marked [71]. The COOH-terminal domain of pore-forming colicins is responsible for the channel formation [15,21,42,43]. This domain of about 20 kDa in general can be easily isolated by limited proteolyric digestion and by itself can fo..rm ion channels in planar lipid bilayers. For colicin A, this protein domain has been crystallized and its three-dimensional structure at 2.7 A resolution should be available in the near

!

future [72]. However this will be the water-soluble structure, since the protein has been crystallized from 2.3 M ammonium sulphate aqueous buffer, and we also would like to know the structure after insertion into the membrane. The dependence on p H of the pore formed by either colicin A or its C-terminal 20 kDa fragment has been measured [40]. The single-channel conductance of the pore formed by colicin A and the fragment increases with pH with an apparent p K of 5.8. At pH 5.0 the gating of both channels are identical and quite similar to those formed by colicin El. However, it was shown that colicin A and not its C-terminal fragment undergoes a pHdependent transition between an 'acidic' and a 'basic' form of the pore with an apparent p K of 5.3. These results suggest that there is a pH-dependent association between the C-terminal domain carrying the lumen of the pore and another do-

I '

I :

s,A

•i

IIJ'1[ t tl ,~11

F'

l! , rrr-,.~

~w"r~',

I

, J ..J_ •r .

-

.it rl

,

.............................

.....

.............

i

?o

i ['

[ f , V ...... ....

Fig. 9. Stepwisecurrent increase after injectionof colicin A (0.1 ng/ml). Soybean phospholipidplanar bilayars separating two aqueous compartments(I0 mM Tris-acetatebuffer(pH 7.0) containing 1 M KCI, 5 mM CaCI2) were formedby transformaU~n¢f fiposomesto planarbilayersas described[21].A voltageof +90 mV was appliedbeforethe additionof coficinA; after the insertiol~ of eight channels,the applied voltage was reversed.After a few seconds, the current reached the bare membranecurrent value. Reversingthe voltageto + 90 mV inducedthe openingof the pores; the appfiedvoltagewas then reversedagain.

459

V

-'I HELI 5X~ , 520-546

Fig. 10. Hypothetical .model fo~ the arrangement of colicin A polypeplide in the lipid bilayer. The residue numbers correspond to tho.~ given in Ref. 39. The pu[ative heli~es arc indicate4. The lining of positively charged residues in helix 4 and of small uncharged hydrophilic residues in hcllces 5 ~md 6 are indicated. One letter symbols are used to represent the amino add sequence.

46o main of the molecule which affects the pore sensitivity to membrane potential. As discussed above, other experimental evidence supports this idea of a probable interaction between the 'rece,ptor' domain and the 'pore' domain.

VIII-B. Modelling of the channel structure Different mo~els have been proposed for the channels formed by pore-forming colicins [15,45,73]. It has also been proposed that only part of the polypeptide is stably inserted in the bilayer, the rest of the polypeptide chain Pipping in and out of the membrane, thereby providing voltagedependent gating for the channel :74]. Circular dichroism studies indieatcd that the COOH-terminal domain contains a higfi proportion of a-helical structure [15,45]. By analogy with the structures proposed for other ion channels or membrane proteins [75-77], this suggests that the pore might be formed as a bundle of a-helices crossing the bilayer. Such helices should be either hydrophobic or amphipatic, having their non-polar faces oriented toward the lipid and their polar faces oriented toward the aqueous lumen of the pore. By using secondary structure prediction methods, six a-helices have been predicted in the COOH-terminal domain of coficins A, E1 and Ib and, among these putative helices, only the three COOH-terminal ones appear to be able to span the membrane according to the hydrophobic moment method of Eisenberg [45]. With the recent appearance of three new colicin sequences, this model has been slightly revised (Fig. 10). Based on conductance measurements, the diameter of the pores is at least 8 A [71] and one can calculate that at least 6 a-helices are required to foFm such a pore. It has thus been proposed that the pore may be formed by a dimer or a trimer of colicins A, E1 or Ib [45]. Studies using site-directed mutagenesis, which allowed the isolation of 45 different mutants in the COOH-terminal domain of colicin A [36] supported the idea that only three helices span the bilayer (helices 4, 5 and 6), but others belong to the mouth of the pore (helices 2 and 3). The recent determination of the minimal length of the C-terminal peptide able to form channels [78] does not rule out the three helices model.

Some mutations led to alterations of the voltage-dependent channel properties in terms of conductance, stability, voltage switching, etc. [36]. These mutants will prove very useful since we can now begin to relate defined amino acid modifications with defined pore properties. Similar studies have been started with colicin E1 [79]. The model present~ .;-n Fig. 10 presents some interesting features. Helix 6, although completely uncharged, shows a remarkable alignment of amino acid residues containing small uncharged side-chains and helix 5 contains an alignment of OH-containing residues. Helix 4 in the class 1 colicins is quite interesting. Its pseudoalignmeut of positively charged lysine residues Js quite reminiscent of the $4 helix of the transmembrane domains of the Na + channel and Ca + channel [80,81]. This positively chat'ged $4 helix was proposed as the voltage sensor of these channels. Site-directed mutagenesis was used for the preparation of a heavy-atom isomorphous derivative for the pore-forming fragment of colicin A. The mutants were designed on the basis of the predicted structural model (Fig. 10), to have the new groups exposed and to leave the pore-forming function unaffected. A successful mutant had a serine replaced by a cysteine (the putative reactive group) and a repeat decapeptide inserted just before it. A mercury derivative led to a 4 A resolution electron density map which clearly shows the oudiue of the protein which appears to be mostly a-hefieal (Tucker, A., Pattus, F., Parker, M., Baty, D., Lazdunski, C. and Tsernoglou, D., unpubfished data).

VIII-C. Molecularity of the colicin channel The three helix model as well as the determination of the minimum length peptide with channel activities imply that the channel is formed by an ofigomer of colicin molecules. However, there is experimental evidence against this hypothesis. In vivo studies [82] suggested a molecularity of 1 for colicin E1 and planar bilayer experiments that examine conductance as iunction of concentration indicate a molecularity of 1 for colicin A [68]. However true kinetics of cell death [83] have not been applied to coficin E1 and the difficulties in obtaining steady-sta~econductance levels in planar

461 bilayers may invalidate these studies. More recently, Bruggeman and Kayalar [84], in an elegant study, determined the molecularity of the colicin E1 channel by stopped-flow ion fluxes kinetics. Although these data are quite convincing, the ion fluxes were determined in vesicles without membrane potential. It was shown that the dependence on membrane potential of coficin E1 depends on vesicle size [85]. This dependence on vesicle size was interpreted as the effect of the aspecifie leak produced by the insertion of colicin E1 in the vesicles. The molecularity of 1 could then be :he molecularity of the lipid bilayer perturbation by colicin monomers inserting within the membrane and not the molecularity of the channel itself. Although colicin A, bound to micelles of an analog of phosphatidylcholine, is monomerie [461, it oligomerises in the presence of negatively charged phospholipids (Pattus, F., t, lpublished resul0. IX, Coliein hnmunity Immunity to channel-forming colicins is provided by polypeptides of 11-18 kDa that span the cytoplasmic membrane three to four times (Fig. 11) [86-881. They specifically interact with the COOHterminal domains of colicins and they confer upon cells protection against the colicin they produce, but not against heterologous colicins with identical modes of action [2]. It is important to point out that they are required to protect the cells against the effects of external colicin, presumably because colicin in the cytoplasm of the colicino-

genic cell cannot act correctly, since the polarity of the transmembrane energy potential is opposite to that required [87]. The amino terminal region of the immunity protein, which is in the cytoplasm, is neither required for insertion nor for function. This has been demonstrated by substituting the first 13 amino acid residues of the immunity protein by the 170 NH:-terminal residues of colicin A. The expresston o( :he hybrid protein obtained is inducible and confers ~rotection to otherwise sensitive cells [89]. The topology of the polypep:;.de chain has recently been confirmed using recombinant D N A techniques. In this technique alkafine phosphatase is fused to defined positions in the gone encoding a membrane protein. Bacteria producing fusion proteins that have alkaline phosphatase a~:fivity are detected using a chromogenie substrate. Since alkaline phosphatase is only active when translocated to the periplasm the alkaline phosphatase moiety of each enzymatically active fusion protein was on the periplasmic side of the membrane. This provides a method for identifying the regions of a membrane protein that are exposed on the periplasmic sicie of the cytoplasmic membrane. Fusions of alkaline phosphatase to sites located in the two putative periplasmic loops and in the putative cytoplasmic loop have fully confirmed the proposed topology for the immunity protein (Geli, V., Baty, D. and Lazdunski, C., unpublished results). Site-directed mutagenesis studies have also been carded out. Mutat/or.s in ',he second pe:'iplasmic loop kave a dramatic affect on the activity of Cai

Fig. 11. Topologyof the coficinA immunitypolypeptidechain in the cytoplasmicmembrane. This topologyhas been probed using various techniques[87,89](Geli,V., Baty, D. and Lazdunski,C., unpublished results).

462 whereas mutations of the same nature in the internal loop have much less effect. The introduction of polar amino add residues in transmembrane hydrophobic regions prevent the insertion of the protein into the membrane. A deletion removing the first membrane-spanning region has the same effect (Geli, V., Baty, D. and Lazdunski, C., unpublished results). Since the immunity protein has a defined orientation, is easily assayable and since we have now constructed recombinant plasmids encoding the immunity protein under the control of inducible promoters [89], this system is suitable for studying the mechanism and the energeties of polypeptide insertion into the cytoplasmic membrane, the role of accessory proteins such as seeA and prlA [90], the energetic requirements, etc. Such studies are in progress. X. Summary The study of colicin release from producing cells has revealed a novel mechanism of secretion. Instead of a buiit-in 'tag', such as a signal peptide containing information for secretion, the mechamsm employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the eoliein as well as other proteins. On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles. They first interact with receptors located at the surface of the outer membrane and are then transferred across the cell envelope in a process that requires energy and depends upon accessory proteins (TolA, TolB, TolC, TolQ, TolR) which might play a role similar to that of the secretory apparatus of eukaryotic and prokaryotic cells. At this point, the type of eoliein described in this review interacts specifically with the inner membrane to form an ion channel. The pore-formh~g colicins are isolated as soluble proteins and yet insert spontaneously into lipid bilayers. The three-dimensional structures of some of these colicins should soon become available and site-directed mutagenesis studies have now provided a large number of modified poly-

peptides. Their use in model systems, particularly those in which the role of transmembrane potential can be tested for polypeptide insertion and ionic channel gating, constitutes a powerful handle with which to improve our understanding of the dynamics of protein insertion into and across membranes and the molecular basis of membrane excitability. In addition, their immunity proteins, which exist only in one state (membrane-inserted) will also contribute to such an understanding. Acknowledgements This work was supported by the CNRS, the INSERM ( e R E No. 861020), the direction des Recherches Etudes et Techniques and the Fondatioga pour la Recherche M&licale. We would like to thank EMBO for providing fellowships to some of us. We are grateful to M. Payan for preparing the manuscript.

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