Topology of Phage X Receptor Protein - The Journal of Biological

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Vol. 259, No. 12, lasue of June 25, pp. 7570-7576 1984 Printed in ir.S.A.

Topology of Phage X Receptor Protein MAPPING TARGETS OF PROTEOLYTIC CLEAVAGE IN RELATION TO BINDING SITES FOR PHAGE OR MONOCLONAL ANTIBODIES* (Received for publication, October 13,1983)

Sergio SchenkmanSQl, AkiraTsugitaQ,Maxime SchwartzS, and Jurg P. RosenbuschQ From the $Unite de Gew2tique Mokculaire, Znstitut Pasteur, 75724 Paris Ceder 15, France and the $European Molecular Biology LaboratocJ, 6900 Heidelberg, FederalRepublic of Germany

Phage X receptor protein of Escherichia coli (LamB structure (7,8),distinctly different from the a-helical configprotein or maltoporin)was purified in a mild detergent uration in bacteriorhodopsin (9),but very similar to the and subjected to prolonged proteolysis byeither tryp- secondary structure of porin (10,11). Several lines of evidence sin or subtilisin. Cleavage occurred at a limited number suggest that portions of the LamB protein are exposed on of sites without affecting the trimeric structureof the both faces of the outer membrane. At the outer surface, LamB protein. Fragments could be dissociated only by heat- protein acts as a receptor for phages such as X and K10 (1, ing in sodium dodecylsulfate to 100 “C. The positions 12); it also binds maltodextrins (13)as well as antibodies (14). of purified fragmentswere determined with respect to At the inner face of the membrane, LamB protein interacts theuncleaved 421-residue polypeptidebychemical analyses. The regions containingtarget sites were with maltose binding protein (3, 5, 15), with the peptidoglymapped aroundresidues 159,203,245, and 370. Based can-lipoprotein complex (16), and with specific antibodies on kinetics of appearance of the different peptides, (17). Basically, three approaches have been used to localize early cleavage events occurred at sites near residues these exposed regions. Point mutations inthe lamB gene have been selected which render bacteria resistant to phages X or 159,203, and 245 and could be distinguished from late events around residue370. Information regarding the K10 (18, 19). These mutations have been mapped by DNA topological orientation of the cleavage sites could be sequencing. It was proposed that the amino acid residues are directly in phage binding obtained from the effect of in vitro proteolysis on the modified in the mutants involved ability of the protein to bind phage X or monoclonal and are thus likely to be exposed at the surface of the cell. antibodies. Loss of phage X neutralizing activity coin- The second approach involved the isolation of monoclonal cided with early cleavage events, whereas loss of an- antibodies directed against the LamB protein (14, 17, 20). As tigenic determinants, known tobe exposed at the cell a result of this approach, it has been demonstrated that a surface, appearedlate. Cleavage regions are thus likely region located within the 70 carboxyl-terminal residues of the to be exposed at thecell surface, a conclusion compat- polypeptide is exposed at the cell surface, since it contains ible with the location of mutationsaffecting the inter- antigenic determinants which are accessible in whole cells.In action of LamB protein with phage in vivo. the present report, we have complemented these approaches by limited proteolysis. Proteases have been shown previously to abolish the phage-neutralizing capacity of the LamB protein (I),while cleavageby subtilisin could be prevented in the Phage X receptor is an outer membrane protein of Escherichia coli encoded by the lamB gene (1, 2), hence also called presence of monoclonal antibodies known to recognize surface epitopes of the phage receptor (17). Using subtilisin and LamB protein. It forms pores which allow nonspecific pertrypsin cleavage, we have studied the kinetics of appearance meation oflow molecular mass molecules (less than 600 and disappearance of the various fragments and the correladaltons) but which display a distinct specificity for maltose and also facilitate the diffusion of maltodextrins containing tion between cleavage events and loss of phage or antibody as many as 7 or 8 glucose residues (3-5). It is therefore also binding ability. The results, in conjunction with earlier data, called maltoporin (5). In thenative state, thephage X receptor indicate that apparently most of the targets for proteolytic cleavage in vitro correspond to regions of the molecule which is an oligomeric protein composed of three identical subunits ( 6 4 , each containing 421 amino acids (6). The amino acid are exposed at, or close to, the cell surface. This conclusion is sequence, inferred from the nucleotide sequence, does not consistent with results obtained using the i n uiuo approaches contain long hydrophobic portions. Spectroscopic evidence mentioned above. indicates a predominance of &pleated sheets in itssecondary MATERIALS ANDMETHODS

* This work waspartly supported by Grant LA 270 from the Centre National de la Recherche Scientifique and by Grant 82 V1279 from the Ministere de 1’Industrie et de la Recherche. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ll Recipient of a postdoctoral fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Technologico (CNPq-Brazil). Present address, Department of Micro, Immuno and Parasitology, Escola Paulista de Medicina, Rua Botucatu 862, 04023 Sao Paulo, Brazil.

Phage X receptor protein was prepared from strain pop1130 (ompR, his) according to procedures described previously (8, 17). Reversible binding of LamB protein to phage X was measured by the protection it conferred to XVh+ against inactivation by a cell extract from E. coli strain 6371 (21). Irreversible inactivation of XVho ( 1 ) and XVLht (22) was followed using the method described (1).With XVLhL, E. coli C600 was usedas indicator strain. Trypsin and subtilisin hydrolyses were carried out in the presence of polydisperse octyl oligooxyethylene over prolonged periods of time (up to 72 h). The detailed procedures used for limited proteolysis, assignment of peptides in the sequence by amino acid composition, and NHT and carboxyl-terminal sequencing were routine peptide chemical analyses which are pre-

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Topology of Phage X Receptor Protein

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cident with the disap~arance of undegraded LamB polypeptide. The kinetics of appearance of the NH2-terminalpeptides, S17.5 and S18, also show a uniform rise. Their cumulative amounts seem much lower than those of the carboxyl-terminal peptides, but this is deceptive (see Fig. 4 in Miniprint) and is due to the irregular distribution of sulfur-containing amino acids within the LamB protein (of 12 methionyl and 2 cysteiRESULTS nyl residues in the wholemolecule, 1 methionine and 2 Phage X receptor protein was treated with either subtilisin cysteines are in thefirst 160 residues). Evaluation of the data or trypsin for 48 h in thepresence of octyl-POE*and analyzed in Fig. 2A shows that the time at which 50% of the final bygel electrophoresis in SDS. The patterns, visualized by amount was reached is also on the order of 12 h. Cleavage Coomassie blue, are shown in Fig. 1 (right).Apparent molec- between residues 150 and 160, like that around 245, also ular weights were assigned to the various cleavage products occursearly. Tryptic fragments (Fig. 2B) can beanalyzed according to their electrophoretic mobility. Individual pep- similarly. The large NHz-terminal peptide T24 is released tides were purified by means of SDS gels, followed by excision first, the half-time of appearance being in this instance 8-10 of appropriate stained areas. Their purity was assessed by a h. In view of their localization, the carboxyl-terminal peptide subsequent SDS-gel el~trophoresis(cfi Fig. 3 in ~ i n i p r ~ t ) TI9 . and its derivatives T18.5, T18, T16, and T15.7 may be The location of these peptides relative to the intact LamB considered together. The half-time of appearance of this group polypeptide is shown in Fig. 1 (left).The results of the of peptides is 10-12 h (Fig. 2B). Both the events at residue following four approaches, obtained for each of these peptides, 203 and around 245 may therefore be considered to occur allowed their localization: (i) apparent molecular weight; (ii) early. The cleavage at 203 seems to occur slightly earlier than amino acid composition; (iii) NHz-terminal sequencing by that around 245, but the significance of this result is difficult Edman degradation; and (iv) carboxyl-terminal sequencing to assess. Based on location and hence on the origin of these using carboxypeptidases A and B. Most of the peptides could peptides, as well as their kinetics of appearance, the events be placed in unequivocal positions based on these criteria. occurring between residues 369 and 392 for both tryptic and The NH~-terminal residues of four tryptic peptides (T15.7 to subtilisin hydrolysis appear to occur late. Early cleavage T19) originating from the carboxyl-terminal domain of the events are indicated as solid arrows at the top of Fig. 1 and polypeptide could not be determined. This is most likely due late ones by an open arrow. to the occurrence, during the long incubation periods used The trimeric state of maltoporin, its overall hydrodynamic (up to 72 h), of latetryptic cleavage events or to minor properties, and its composition were not detectably modified contamination with chymotryptic activity (23). The subtilisin after 48 h of enzymatic hydrolysis. This conclusion is based fragment S19, which has a unique carboxyl-terminal end, was on three lines of evidence. Conventional sucrose gradient slightly heterogeneous in size, indicating, in this instance, a low selectivity of cleavageby this enzyme (24). All other centri~gationin octyl-POE gave vastly different sedimentaresults from conventional peptide chemistry were quite une- tion coefficients for the un~ssociatedtrimer, as opposed to the dissociated and denatured monomer (8), reminiscent of quivocal. . ~ sedimentation coefThe kinetics of appearance and disappearance of the var- analogous phenomena with p ~ r i n The ious peptides (shown in Fig. 1) distinguished early from late ficient observed after proteolysis was indistinguishable from events. The disappearance of the intact polypeptide and the that of the native trimer. Immunoprecipitation with monocloappearance of fragments were monitored using 35S-labeled nal antibodies precipitates all fragments quantitatively, thus of the subprotein (Fig. 2). A parallel analysis was performed using demonstrating that content and tertiary structure unit are not grossly altered. Finally, maltoporin (as porin) unlabeled protein with q u a n t i ~ t i o nof Coomassie blue eluted from stained bands (see Miniprint). The results are shown runs as a trimeric unit on SDS-gel e~ectrophoresis, asit is for subtilisin and trypsin in Fig. 2, A and B, respectively. dissociated only by heating above 90 “C. Although all these Analysis of A shows that the cumulative amounts of the criteria contributelittle information asto whether small carboxyl-terminal peptides S19, S14, and S13 rose uniformly. conformational changes had occurred, they firmly show that The time at which 50% of the final value was reached (12 h) the trimeric structure is maintained. The experiments describedhavebeenperformedwith coincided with the timeat which 50%of the intactpolypeptide LamB in solution. In order to obtain information on the had disappeared. Of the peptides, ,519 is the first to appear while 513 and 514 clearly lag behind. From the net decline of locations of the cleavage sites relative to the binding sites of S19 after 24 h, and the complementary increase of S13 and phages (presumed to be at the outer face of the membrane), S14 thereafter, it appears likely that the latter two are derived or of antigenic determinants (located on either side of the from S19. This is in agreement with the location of these membrane), we have investigated the effect of proteolysis peptides (Fig. 1) also. Cleavage around residue 245 (for the both on the ability of LamB protein to neutralize phage XVh, imprecision, cf. Miniprint) is therefore an early event, coin- and of monoclonal antibodies to precipitate the protein (Fig. 2, C and D).The loss of phage-neutralizing activity followed ’ Portions of this paper (including“Experimental Procedures,” partvery closelythe kinetics of the early cleavage events, as seen of “Results,” part of “Discussion,” Figs. 3-5, and Table I) are pre- by comparing C to A and D to B in Fig. 2. From that figure it sented in miniprint at the end of this paper. Miniprint is easily read is seen that the decrease of phage inactivation capacity conwith the aid of a standard magnifying glass. Full size photocopies are curred with the disappearance of the undegraded polypeptide available from the Journal of Biological Chemistry, 9650 Rockville band. These results therefore demonstrate that early cleavage Pike, Bethesda, MD 20814. Request Document No. 83M-2943, cite events destroy the A receptor activity of LamB protein. When the authors, and include a check or money order for $3.20 per set of photocopies. Full size photocopies are also included in the microfilm the effects of proteolysis on epitopes were analyzed, those located in vivo at the inner surface of the outer membrane (cf. edition of the Journal that is available from WaverlyPress. The abbreviationsused are: octyl-POE, polydisperse octyl oli- sqwlres in Fig. 2, C and D)appeared unaffected by proteolytic

sented in detail in Miniprint at the end of this paper.’ M o n ~ l o n a l antibody preparation and immunological reactions were performed as described (14). The antibodies were purified fromascites fluids by protein A-Sepharose4B chromatography (17). For antibody precipitation, 35S-labeledLamB protein was diluted in 0.5 ml of 2% Triton x-100 and 50 mM Tris-HC1, pH 8.2. For details, see the Miniprint.’

gooxyethylene; SDS, sodium dodecyl sulfate; PPO, 2,5-&phenyloxazole; POPOP, l,4-b~s[Z-(5-phenyloxazolyl)Jbenzene.

J. P. Rosenbusch, unpublish~results.

Topology of Phage XProtein Receptor

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FIG. 1. Proposed map of proteolytic cleavage sites on the phage X receptor protein and the resulting peptide fragments. The patternsobtained by polyacrylamide gel electrophoresis in SDS (right) show the cleavage products obtained after incubating purified LamB protein for 48 h in the presence of either subtilisin (left l a n e ) or trypsin (rightlane). The central slot contained5 pg of untreated LamB protein. The gel was stained with Coomassie blue. Proteolytic fragments obtained after subtilisin and trypsin treatment are marked S and T, respectively, followed by a number which corresponds to their apparent mass in kilodaltons. All numbered peptides were purified. Analysis of their amino acid composition and NHz- and COOH-terminal sequences are presented in the miniprint. Minor tryptic peptides or fragments occurring only transiently were not analyzed. The combined results of these analyses are shown as linear sequences in this figure. The heavy horizontal line (top) corresponds to the undegraded 421-residue LamB polypeptide. Horizontal lines below indicate the location of the cleavage products. Numbers at the end of these lines correspond to the positions of the NHz- and COOH-terminal residues of the peptides. The series of dots at theNH2 termini of peptides S19 and T15.7 to T19 indicatesthat theexact positions of their ends are not known (see miniprint). The solid arrows at the top of the figure indicate the regions where cleavage events are presumably primary, while the open arrow is likely to correspond to secondary cleavage sites (see text and Fig. 2). The notations S or T above the arrows indicate whether subtilisin or trypsin cleaves in the corresponding region. The notation (S) indicates that subtilisin cleaves partially in the corresponding region (between residues 204 and 205) and that thetime course of appearance and disappearance of the 3 peptides (S3, S4, S8) could not be determined. Filled circles indicate regions where amino acid changes have been found in mutants resistant to phage X, K10, or both (18, 19).5

cleavage of the protein, as theirimmunoprecipitation was not markedly impaired. Epitopes located in the cell at theexterior surface of the outer membrane clearly lost their antibody binding ability with progressing proteolysis. Analyzing the kinetics of appearance and disappearance of the various proteolytic fragments, it is obvious that the decrease of immunoprecipitation did not correlate with early cleavage events, but that itmay be correlated to the increase of S13 andS14, and correspondingly, to the disappearance of subtilisin fragment S19. It thus appears related to a late cleavage event. This suggests a location of the corresponding epitopes for subtilisin hydrolysis between residues 379 and 421, or at least that a region comprising 42 residues at the COOH-terminal end of the protein contributes significantly to the conformation of the epitopes. A corresponding analysis for tryptic hydrolysis yields a similar, although less significant correlation, between the appearance of the sum of fragments “15.7 and T16.5 and the decrease of immunoprecipitation due to exterior epitopes. This would indicate that a region essential for the function of the externalepitopes is located within the last 30 residues of the X receptor protein.

DISCUSSION

Correlating structure and function in a membrane protein requires knowledge of its topology relative to the membrane. We have used limited proteolysis to approach this question with phage X receptor, an outer membrane protein of E. coli. Subtilisin and trypsin cleaved the polypeptide into 8 and 7 major fragments, respectively. Using standard chemical methods, all of these peptides could be assigned unequivocally in the sequence of the protein (cf. Fig. 1 and Footnote 4). Where uncertainties arose, such as assigning unequivocal NH2-terminal residues to tryptic peptides in the carboxyl-terminal domain of the protein, they have no significant impact on the localization of these peptides. Studying the kinetics of appearance and disappearance of the different peptides substantiated the location of the various fragments. Thus, as the initial subtilisin peptide S19 started to disappear after 24 h, ‘Recently, we have assigned peptides on the basis of size and amino acid composition by a computer program, using a window sliding over the entire polypeptide chain in search of the best fit. Excellent agreement with the results presented here was obtained (unpublished results).

Topology of Phage h Receptor Protein

TlMElhrl

FIG. 2. Kinetics of proteolytic events and their correlation with theloss of functional sites. "S-labeled detergent-solubilized LamB protein (10 pglml) was treated with subtilisin ( A and C) or trypsin ( B and D).In thetop panels, the disappearance of the LamB polypeptide and the appearance of various cleavage products were followed by performing SDS-polyacrylamide gel electrophoresis and measuring the radioactivity present in the corresponding bands. All results are expressed in per cent of the value obtained at time 0 with intact LamB. Symbols in A: undegraded protein (O), S19 + S14 + ,313 (O),S19 (V), S18 + s17.5 (A), S14 + S13 (A);B: intact protein (O),T19 + T18.5 + "18 + T16 (O),T24 (A), T16 + T15.7 (A).In C and D, the ability of the LamB protein to inactivate XVh (V)(cf. ordinate on the left) or its immunoprecipitation (right coordinate), by monoclonal antibodies directed toward epitopes on the inner surface of the outer membrane 436 (0)or 141 (B)or its outer surface 302 (VI or 72 (X) are shown as a function of the duration of proteolysis. Ftesults (not shown) obtained with two other monoclonal antibodies which recognize external epitopes (177 and 347) were indistinguishable from those shown here for 302 and 72.

two derivatives thereof, S13 and S14, appeared. In this, as in other cases, it was possible to regard various fragments as members of a setof peptide families, each family corresponding to a given region of the LamB polypeptide. The chemical analyses of the peptides as well asthe kinetics of their appearance allowed definition of four major cleavage regions in the vicinity of residues 159, 203, 245, and 370. All cleavage events occurred very slowly, with half-times between 6 and 40 h, even though high concentrations of proteases were used (25% per weight). The cleavage events around residues 159, 203, and 245 occur early and coincide, within the limitation of the methods, with the disappearance of the intact LamB polypeptide. In contrast, cleavage events occurring around residue 370 took place distinctly later. From the results obtained it was not possible to decide whether these various events occurred independently or were linked together. Since the overall structure of the LamB trimer was not detectably changed even after prolonged proteolysis, it seems likely that early proteolytic target sites are accessible from the solvent without major conformational changes of the protein andthat limited hydrolysis does not cause such changes. The same sites were also accessible whenthe protein was reconstituted in phospholipid vesicles (data not shown). Although proteolysis was even slower in this instance, the peptide patterns, although varying in relative amounts, remained unchanged. If proteolytic cleavage sites were buried within the membrane, or if the protein were incorporated in an anomalous way into tight reconstituted vesicles (261, the pattern of the bands would surely be affected qualitatively, although not quantitatively. This result suggests therefore that asin viuo the cleavage sites arein regions exposedto the solvent rather than being embedded in the lipid bilayer core. Four lines of evidence support this notion. (i) All subtilisin cleavage events have been shown to be prevented in in vitro

7573

experiments by monoclonal antibodies specific for epitopes which are exposed at the cell surface in uiuo, but not by monoclonal antibodies whichrecognize epitopes located at the inner face of the outer membrane (17). (ii) The late cleavage events which occur around residue 370 (between 369 and 392) specifically destroy the ability of LamB protein to bind those monoclonal antibodies which recognize external determinants. In this case, small conformational changes following early cleavage events appear likely, as hydrolysis of peptide bonds in this region occurs significantly later. It has been shown previously that functional epitopes are required for binding of monoclonal antibodies and, more specifically, that 70 residues from the carboxyl-terminal end are required for this binding (20). (iii) Concomitant with the early cleavage events occurring with both enzymes, LamB protein loses the ability to neutralize XVb. Experiments reported in the miniprint suggest that it is the binding of the phage which is affected. In vivo this reaction can be presumed to occur at the cell surface. (iv) Mutations rendering the cells resistant to phages X or K10, or both, were previously shown to result in alterations located exclusively in regions now found to be susceptible to proteolytic cleavage (18,19): The positions of these alterations are shown by dots in Fig. 1. Although none of these observations, taken by themselves, would allow the drawing of unequivocal conclusions, these arguments, taken together, strongly suggest that the cleavage regions are exposed at the cell surface. The evidence considered for each region individually yields the following. The most unequivocal conclusion is obtained for the region around residue 245, which is atarget for early cleavageby both trypsin and subtilisin and which contains sites where alteration by mutation leads to phage resistance. The region around residue 159, which constitutes an early target for cleavage by subtilisin, contains several mutant sites and is thus also likelyto be exposed at the cell surface. The observation that it is not cleaved by trypsin is not surprising since the closest basic residues are Arg,,, and ArgI7,. This may indicate that the exposed loop is rather small. Cleavage at Argm3 bytrypsin is clearly an early event, whereas subtilisin hydrolysis in the same area is only partial (cf. co-existence of peptides S3, S4, and S8). Since no mutant site has yetbeen found in this region, it is conceivable that Argzo3 could be exposed at the periplasmic side of the membrane. Regarding the cleavage region closest to the carboxyl-terminal end of the protein, it was surprising that it is subject to late cleavage events only, since it is related not only to both epitopes accessible at the cell surface, but also contains two mutant sites. This could mean that the polypeptide backbone is present in a particularly taut configuration in this region or that the proteasesensitive bonds are cryptic. The analyses performed by no means rule out small secondary conformational changes resulting from early cleavage events. Such qualifications pertain to all of the approaches used, regardless of the protein and the types of proteases considered. With these reservations in mind, we propose as a working hypothesis that the four regions susceptible to proteolytic cleavage in vitro are exposed at, or in close proximity to, the cell surface in uiuo. It is likely that the polypeptide segments separating the exposed regions are highly structured regions, most of which are embedded in the membrane. This and previous studies indicate that the LamB polypeptide is composed of two segments apparently endowed with very different properties. The NHz-terminalsegment, which has a length of approximately 150 residues, is totally resistant toproteolysis, A. Charbit and M. Hofnung, personal communication.

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behavior reminiscentof the complete resistance of nonspecific 32 porins of the outer membrane of E. coli (10). Moreover, no 10. Rosenhusch, J. P. (1974) J. BwL Chem. 249,8019-8029 alterations by mutations leading to functional defects (phage 11. Garavito, R. M., Jenkins, J., Neuhaus, J.-M., Pugsley, A. P., and Rosenbusch, J. P. (1982) Ann. Microbwl. (Paris) 133A, 37-41 resistance or decreased maltose transport) have been observed 12. h a , M.(1979) J. Bacterkl. 140,680-686 in that region. It thus may be tightly packedwithin the 13. Ferenci, T., Schwentorat, M., Ullrich, S., and Vimart, J. (1980) membrane, exposing few residues at either surface. InterestJ. Bacterwl. 142,521-526 ingly, this segment has been attributed with an important 14. Gabay, J., and Schwartz, M. (1982) J. Bwl. Chem. 2 5 7 , 66276630 role in export and localization of the protein (27-29). The carboxyl-terminal segment, which is approximately 270 amino 15. Bavoil, P., and Nikaido, H. (1981) J. BioL Chem. 266, 1138511388 acids long, contains a limited number of protease-sensitive 16. Gabay, J., and Yasunaka, K. (1980) Eur. J. Biochem. 104, 13sites and alterationsby mutations of several residues leadto 18 defects inphage adsorption(cf. above) or in maltose transport. 17. Schenkman, S., Couture, E., and Schwartz, M. (1983) J.Bacterid 155,1382-1392 A region between residues 230 and 330 contributes critically (6,19,20) to the latter.A number of amino acid residues thus 18. Roa, M.,and Clement, J. M. (1980) FEBS Lett. 121,127-129 appear to be exposed inthis segment which contributes more 19. Clement, J. M., Lepouce, E., Marchal, C., and Hofnung, M. (1983) EMBO J. 2,77-80 directly to thebiologicalpropertiesoftheLamBprotein 20. Gabay, J., Benson, S., and Schwartz, M. (1983) J. Bwl. Chem. assessed. Whether and how the two segments interact to form 258,2410-2414 a single or multiple domain struture remains to be determined. 21. Schwartz, M. (1975) J. MOL BkL 99,185-201 Acknowledgments-We thank A. P. Pugsley, J. Gabay, and H. Shuman for helpful suggestions and discussion; F. Vilbois and C. Jone for technical assistance in amino acid analysis and sequence determinations, and C. Barber for typing the manuscript. We are grateful to A. Charbit and M. Hofnung for communicating unpublished results. Note Added in Proof-The amounts of cysteines have been determined in the peptides (1.6 in S18; 1.6 in S17.5; 0 in S8, S4, S3, S14, 513, and S19; 1.2in T24; and 0 in all other tryptic peptides). Similarly, there are5.1 tryptophanes in S18,5.3 in S17.5,0.5 in S8,O in s4,0.6 in S3,5.5 in $314, 7.8 in S13, 10.6 in S19, 18.2 tryptophanes in LamB protein, 4.8 in T24, 10.8 in T18.5, 9.9 in T18, 8.2 in “16, and 8.2 in T15.7. NH, termini were further determined for S18 (ValAspPhef, T I 9 (GlyLeuSer), and “16 (GlyLeuSer). The NH2 terminus of the undegraded polypeptide is ValAspPheHisGly. The carboxyl-terminal ends beginning with “245” (see Fig. 1) are due to a pseudochymotryptic activity, rather thanchymotryptic c o n ~ i n a n t s . REFERENCES 1. R a n ~ l l - H a z e l ~ u eL., r , and Schwartz, M. (1973) J. Bacterid. 116,1436-1446 2. Schwartz, M. (1983) Methods Enzyml. 97,100-112 3. Wandersman, C., Schwartz, M., and Ferenci, T. (1979) J. Bacterial. 140, 1-13 4. Luckev. M.. and Nikaido., H. (1980) . . Proc. Nut1 Acad. Sci U. S. A. 77,167-171 5. Neuhaus, J. M., Schindler, H., and Rosenbusch, J. P. (1983) EMBO 3. 2,1987-1991 6. CIBment, J. M., and Hofnung, M. (1981) Cell 27,507-514 7. Ishii, J. N., Okajima, Y., and Nakae, T.(1981) FEBS Lett. 1 3 4 , 217-220 8. Neuhaus, J.-M. (1982) P&D. thesis, University of Basel, Switzerland 9. Henderson, R., and Unwin, N. (1975) Nature (Lord.)2 6 7 , 28-

22. Hofnung, M., Jezierska, A., and Braun-Brenton, C. (1976) Mol. & Gen. Genet. 145,207-213 23. Keil, B. (1971) in The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 3, pp. 249-275, Academic Press, New York 24. Markland, F.S., and Smith, E. L. (1971) in The Enzymes (Boyer, P. D., ed) 3rd Ed, Vol. 3, pp. 561-608, Academic Press, New York 25. Osborn, M. J., Gander, J. E., Parisi, E., and Carson, J. (1972) J. Bhl. Chem. 247,3962-3972 26. Grabo, M. (1982) Ph.D. thesis, University of Basel, Switzerland 27. Hall, M.N., Schwartz, M., and Silhavy, T. J. (1982) J. Mol. Bid. 166,93-112 28. Hall, M. N., Gahay, J., and Schwartz, M. (1983) EMBOJ. 2.1519 29. Benson, S. A., and Silhavy, T. J. (1983) Cell 32,1325-1335 30, Wandersman, C., and Schwartz, M. (1982) J. B a c ~ r w L151,15‘33

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31. Ito, K., and Date, T., and Wickner, W. (1980) J. Biol. Chem. 265,2123-2130 32. Laemmli, U. K. (1970) Nature (Lo&.) 227,680-685 33. Suter, P., and Rosenhusch, J. P. (1975) Eur. 3. Bkchem. 64, 293-299 34. Dianoux, A.-C., Vignais, P. V., and Tsugita, A. (1981) FEBS Lett. 130,119-123 35. Tsugita, A., and Scheffler, J. J. (1982) Eur. J. Bioehem. 124, 585-588 36. Isobe, T..Yanapida, M., Boosman,. A.,-. and . Tsurrita. A. (1978) . . J. Moi Bwl. 125,339-356 37. Tsurrita. A.. Blazv.B., Takahashi., M... and Baudras.. A. (1982) . . FgBS Lett. 144; 304-308 38. Tsugita, A., Gregor, I., Kubata, I., and van den Broek, R. (1979) in C y ~ c h r o Oxidase ~e (King, T. E., Orii, Y., Chance, B., and Okunuki, K., eds.) pp. 67-77, EIsevierfNorth Holland Biomedical Press, Amsterdam 39. Hirs, C. H. W. (1967) Methods Enzymol 1 1 , 197-199 40. Maeda, K., Scheffler, J.-J., and Tsugita, A. (1984) ~ o p ~ - S ~ & r ~ 2.PhyswL Chem., in press


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..

.

p a r r pl acl0 t e 0 1 y 9 t1IhSne. c a s e o f ~ ~ u b t l l 1 5 1pnr. o t e o l y S 1 l e5a d s to d c o m p l e t e disappearance o f t h e 4 7 4 0 0 d a l t o n Lam8 p o l y p e p t i cd he aw l nl t h f o r m a t i o n o f p e p t l d c s o f a p p r o x l n a r e l1y9 0 0 01,8 0 0 01.1 5 8 0 . I4000 and 13000 d a l t o n and o f some slnaller p p f l d e s r l t h m o l e c u l a r w e l q h t s r a n q l n gf r o m 8000 t o 3000 IFIqure 351. T h pe sp et l d e s were p u r l f l e d by p r e p a r a tgi ve le e l e c t r o p h o r e s l s . me r c s u l t l n g fractions a r e S h o r n I n t h e same f l q u r e .

7575


7576 T a b l e 1 A m i n o a c i dc o m p o s i t i o na n de n dg r o u pd e t e r m i nt l o n o f s u b t i l i S i n ( S ) a n dt r y p t i c( T )

518 25'50'

TheO. 25'

18.2 18.0 7.5 7.4 8 12.8 12.3 20.4 19.6 3.3 3.5 23.5 23.3 14.0 14.0 5.3 6.0 1.4 1.6 5.6 5.8 7.9 8 7.9 7.6 7.1 8.1 7.9 9 3.3 3.0 1.2 6.8 6.1 5.7

17 14 20 3 20 14 7 1 6 8

3

7 6 2 6

n.d. n.d.

Sll.! 50

n.d. n.d.

. . M U ( t h e o . ) 11606 nu(gel1

4.4 2.8 2.2 4.2 0.2 3.0 2.4 2.1

0.4

0.3 2.0 1.0 1.6 0.5 1.1 0.1

0.9 2.0 0.8 1.6 0.5 1.5 0.8

4 4 4 4 0

3 3 4 1 0 2 1

3 1 3 0

n.d.

...

4194 (4500) 205 147

Lam0 25'50'

61.0 21.1 24.1 41.6 8.2 50.9 48.7 34.5 32.5 11.0 11.3 9.4 9.7 15.1 16.3 19.0 19.0 21.9 21 .O 19.3 19.4 5.7 5.6 18.6 18.1 16.0 15.1 2.1

63.2 22.2 21.3 42.6 8.5

n.d. n.d.

1

6 8

8 8 3 I 6 2 6

611 211 21 42

8 48 33 21 12 19 19 22 20 19 6 16 19

22 I I 10 3 11

1

8 10

4 7 3 2 6

4 0

I

1

7 5 5

1.02 1.9 1 4.4 6 2.5 3 2.1 3

0.8

1

2.7 3 2.7

3

n.d. n.d.

Tt4 50 Thoo.

28.2 10.8 11.9 24.8 4.1 25.2 11.0 9.0 1.0 6.9 12.0 10.0

26.8 10.4 10.6 24.2 11.4 24.1 11.0 9.2 1.2 7.4 12.0 9.3

8.8

9.3

2.4 2.1 1.2 1.2 9.59.8 2n.d.

21 11 14 24

4 24 11 10 2

8 12 12 10 3 I

9 2 6

4 20 13 9 11

4 9 I 2 8 6 0

12

10 3

n.d. n.d.

0 0

1

4 3 1

1

5 3 1

0

4

8.8

8.4

9

6.2 2.1

6.1 2.9 1.9 6.2

I 2

8.4 1.1

n.d. n.d.

0

9

-

Kinetip o c srf o f e o l y s m l ~o n i t o r e d C by oonassie Blue s t a i n . P s p t F S -re a n a l y z e da n d quantitated as i n P i 9 . 2 e, x c e p t h a at b s o r b a n c eo f e l u t e ds t a i n( c f t. e x t a) 6t # 0 run was m o n i t o r e dP. a n e l A. SI8 ( 0 1 1 517.5 1.). 519 ( v ) s l 4 [ A ) S I 3 ( A ) . Heavy l i n ersa p r e s e tnh ce a l c u i a t c d OMS otfhN e - t e r m i n apl e p t i d e s , 518 and 517 ( u p p e r l i n e z o f t hCe - t e r m i n a l p p t i d e s Is19, S I P , 513; lower line). p a n e l 8. T24 I A ) , T18.5 and T l 8 I O 1 I TI9 [ ), and TI6 [ D l . Heavy l i n m c a t e t h e 5Um ot hfCe- t e r m i n a l pcptldt. T19, T18.5, Tl8 and T16 ( u p PlC i n ea]n,t hduen i q uNe- t e r m i n a l p e p t i d e T24. e f f e c t of Lams p r o t e o l y s i s o n p h a g e Z b i n d i n g and neutralization. When p r o t e i n O f E . C o 1 i K12 i s m l r e dw i t h hvh' ( p h a g e Z w i t h a w i l dt y p eh e s f r a n g et h) e v i r U S r e r a i n s t h ae b l l i t y t o I n f e cbta c t e r i a (1). I n Contrast, L ~ O B ;ratein f y several o t h e rv l l d s t r a i n s of Such a s s t r a i n 6311 n e u t r a l i z e s Zvh (21). I t was s h o wpnr e v i o u s f y 1 2 1 ) t h a t even t h o v g h Lam0 p r o t e i nf r o m E.c01i K12 f a i l s t o n e u t r a l i z e ZVh , I td o e 5i n t e r a c tw i t ht h i s phage. This m i b l e b i n d i n g i s d e m o n s t r a t e b d tyh oe b s e r v a t i o tnh a t Lam0 p r o t e i fnr o m E . C O 1 I K12 c a n p r o t e c t Zvh' a g a l n s nt e u t r a l i z a t i o n by t h e Lam0 p r o t e i En . r 6 3 7 1 . The e x p e r i mPeiingt. d5 eAm o n s t r a t h e sa t . Once t r e a t e d w?th L r y p S l n or s u b t i l i s i n f o r 4 8 h r s . , Lam0 p r o t e i n Of E . C O 1 i had l o s t t h ea b i l i t yt op r o t e c t hvh' a g a i n s t inactivation by Lam6 p r o t e i n f E . c d i 6371. m e r e f o r tp, r o t e o l y e i ad e s t r o y e dt h ea b i l i t yo f Lam0 p r o t e i n toThe h i n d i n 4 i s b e l i e v e d( 2 1 ) to b et h ef i c s t o h a a e h I n a r e v e r r l h l e manner. Lam0

B,

8 6 0

12

32.2 31.9 8.9 8.2 8.8 8.2 14.6 14.9 4.0 4.0 20.0 20.1 13.0 13.0 3.9 5.2 6.3 5.1 9.3 10.3 3.5 4.0 10.1 9.1 5.6 5.8 2.2 2.3 7.5 7.5 6.1 6.6 n.d n.d.

20167 (19000)

246 421

I4 4.2 3.1 2.1 3.4 0.0 3.1 2.6 2.1 0.6 0.3 3.6 1.1 3.1 0.9 3.5 0.1

119 I h a o . 25'50'

25'50' 4.3 3.1 2.5 3.4 0.0 4.0 2.5 2.5 0.5 0.3 3.5 1.0 2.6 0.9 3.6 0.1 n.d. n.d.

33 9 9 14

4 20 13 9 11 4 9 I 2 8

b 0

12

26.5 26.8 7.9 7.8 5.9 5.6 11.0 11.0 2.8 3.0 13.6 14.5 9.5 10.1 3.5 4.9 5.6 4.0 8.D 8.9 4.2 4.4 9.0 8.8 3.6 3.2 1.9 2.0 6 . 7 6.8 4.6 5.4 n.d. n.d.

In F i g . 4 , t h e r e s u l t s o f experiments Kinetics of peptide apparance. In t h e s e e x p e r i m e n t s ,t h e t h o s e shown In Pig.2Aand 0 a r e Shown. a n a l o g o u st o b yd e l C r m i n l n gt h e k i n e t i c s were S t u d i e db yf o l l o w i n gt h ei n t e n s i t i e so fb a n d s e l u t e dC o o m a s s i eB l u es t a i n . The f o l l o w i n gp o i n t s may b en o t e d . ( I ) I t can b e seen from Panel A t h a t h ek i n e t i c s of appearanco e ifn d i v i d u apl e p t i d e s is f u l lcyo m p a t i b lwe i tthh e igrr o u p i nign t o families which r e s u l t I from t h e c h e m i c aa ln a l y s i sl l. i ) T ha ep p e a r a n co t hef e two amlno t e r m i n ap le p t i d e s (517.5 and 518) i s seen more c l e a r l y u s i n g an u n s p e c i fpi cr o t e5i nt a L n I C o ~ m a s s i eB l u e )t h a nb yl a b e l l l n qw l t hs u l f u rc o n t a i n i n g amino a c i d sr h l c h a c e i r r e q u l a r l y distributed-in t h ep o l y p e p t i d e .

0

30.6 28.6 33 8.1 9.1 9 10.4 9.1 9 15.0 16.2 14 4.4 1 4 20.8 22.4 19 13.0 13.0 13 4.1 5.1 7 5 9 6.56.2 9.4 9.4 1 1 4 5.1 5.0

19.1 19.3 20 6.11 6.2 7 4.1 4.5 5 9.0 9.0 9 2.8 2.9 3 10.6 11.2 10 6.2 6.2 6 3.1 4.2 10 5.3 5.2 1.5 8.0 3.0 3.0 3 6.3 6.2 I 0.8 0.8 1 2.0 1.9 2 5.3 5.7 6 4.7 4.6 5 n.d. n.d.

0 4 1

4890 (5000) 162

4

4 4

4 0

4 3 4 1 0 3 1 3 1

4 0 0 1

4550 (4500) 204 244

202

33 9 9 14

8.9 9.5 2.8 2.6 0.0 0.0 4.2 4.2 0.9 0.9 4.3 4.5 3.0 3.0 2.3 2.5 0.8 0.8 0.9 1.0 5.0 5.2 2.6 2.4 1.4 1.0 0.0 0.0 0.0 0.0 4.2 4.6

0

13103 (13500) 263 318

22569 (24000)

to

n.d. 0.d.

4.1

9 5 4

. .

n.d.

MY(thoa.) 41400 HW(gs1) ~410001 From 1

32.9 32.1 9.08.9 8.0 1.0 14.0 13.9 4.3 3.9 19.6 20.1 13.0 13.0 4.8 5.4 7.91.2 9.9 10.6 3.7 3.9 9.2 9.2 6.56.9 2.1 2.2 7.2 7.a 5.8 6.5

T h e o . 25s450, Theo.

8.9 4.4 3-2 7.5I 0.7 1.5 5.2

1319

14393 (14000) 246 769

TheO. 25'

n.d.

1

21.5 22.2 6.9 6.4 6.6 5.1 9.9 9.9 3.0 3.1 11.6 12.1 1.0 1.0 4.6 5.0 7.4 5.4 8.0 8.3 3.5 4.4 6.9 6.1 1.4 1.8 2.0 1.9 5.6 5 . 9 4.6 4.6

0 1

n.d.

nutthso.) nu(8el) PP*.

12 20 3 20 13 I

S8 25'50' 9.9 5.0 3.5 7.5 0.8 7.1 5.1 3.7 1.1 1.9 4.1 2.8 2.2 0.7 2.9 2.7

( 8000)

4.3 2.6 2.1 3.7 0.0 3.3 2.3 2.3

0.7

15

17012

(lye,

F""

Theo.

14.9 15.4 7.5 1.3 10.1 10.1 19.5 20.1 3.0 3.0 20.5 20.7 13.0 13.0 6.5 5.7 0.8 0.1 4.8 5.3 1.6 7.1 7.6 7.3 7.5 7.2 2.8 2.2 6.5 6.3 6.2 5.6

Theto.

32.2 1.3 8.2 13.1 3.4 21.0 13.0 5.6 1.5 9.4 10.2 4.0 4.1 8.7 8.4 6.1 6.1 2.6 2.5 7.5 7.9 5.86.26

33 9

n.d. n.d.

0 12

30.2 1.1 9.7 13.1 3.6 20.8 13.0 8.6 1.5

9 14

4 20 13 I

9 11

4 9 I 2

8

20224 (190001 245 421

29

8 7 11 3 15 10 6

8 10 4 9

4 2 7 5 0

9

26.5 1.3 6.9 11.0 3.3 16.1 10.1 4.9 5.0 9.2 4.2 8.9 8.6 2.6 2.1 2.0 2.3 6.1 6.8 4.7 5.1

26.5 1.4 7.1 11.0 3.5 15.0 9.0 3.3 4.4 7.1 4.1

n.d. n.d.

28

8 7 11 3 14 10 6 8 10 4 9 3 2 6 5 0 9