Homology Model of Surface Antigen OmpC F 10449

 

J ournal ournal of Biomolecular Structure &  Dynamics, ISSN 0739-1102 0739-1102 Volume 18, Issue Number 2, (2000) ©Adenine Press (2000)

Homology Model of Surface Antigen OmpC From Salmonella typhi and its Functional Implications http://www.adeninepress.com  Abstract

Homology based 3D structural model of the immunodominant major surface antigen OmpC from Salmonella typhi, an obligatory human pathogen, was built to understand the possible unique conformational features of its antigenic loops with respect to other o ther immunologically cross reacting porins. The homology model was built based on the known crystal structures of the E. coli porins OmpF and PhoE. Structure based sequence alignment helped to define the structurally conserved regions (SCRs). The SCR regions of OmpC were modelled using the coordinates of corresponding regions from reference proteins. Surface exposed variable regions were modelled based on the sequence similarity and loop search in PDB. Structural refinement based on symmetry restrained energy minimization resulted in an agreeable model for the trimer of OmpC. The resulting model was compared with other porin structures, having b-barrel fold with 16 transmembrane β-strands, and found that the variable regions are unique in terms of sequence and structure. A ranking of the loops taking into account the antigenic index, the sequence variability, the surface accessibility in the context of the trimer, and the structural variability suggests that loop 4 (151-172), loop 5 (194-218) and loop 6 (237-264) are the best ranked B-cell epitopes. The model provides possible explanations for the functional and unique immunological properties associated with the surface exposed regions and outlines the implications for structure based experimental design.

A. Arockiasamy and S. Krishnaswamy*

Bioinformatics Centre, Department of Genetic Engineering, School of Biotechnology Biotechnology,, Madurai Kamaraj University University,, Madurai-625 021, India

 Introduction

Porins are pore forming proteins present as trimers on the outer membrane of Gram negative bacteria which facilitate intake and disposal of nutrients and waste materials of size <600 Da respectively. These water filled channels allow only hydrophilic solutes to passporin through the (2) pore usingepitopes monoclonal antibodies raised against native trimers as (1). well Studies as synthetic showed that porins are highly cross-reactive and the crucial domains are conserved (3). Porins are shown to be B-cell polyclonal activators (4) and also shown to induce cytokines in cultured human monocytes (5). Also porins have attracted much attention, as they are potential cell surface antigens, which can display heterologus sequences on the bacterial cell surface (6) towards the development of oral vaccines (7). Defence molecules like lactoferrins and C1q, a component of complement system, were shown to bind to enterobacterial porins (8,9). Salmonella porins were found to induce both humoral and cell mediated immunity in animal models (10,11). A recent study demonstrated that outer membrane porin could induce apoptosis in epithelial cell line SVC1 (12). The surface exposed regions of these outer membrane proteins are believed to play a crucial role in the survival of the bacterial pathogens inside the host system (13). Results from these studies suggested us that investigations of variable surface exposed regions of porins might help in understanding the diverse role of these proteins in biology, especially in host interaction, pathogenesis and immunology. Crystal structures of few porins (14,15) from  E. coli and other bacteria show a similar three-dimensional architecture. Enterobacterial porins show a high degree of homology

*Phone: +91-452-859141; Fax: +91-452-859105; E-mail: [email protected]. kr [email protected] in

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262  Arockiasamy and Krishnaswamy 

in terms of sequence (16) and structure (17). General diffusion pores like OmpF and PhoE have a 16 stranded β-barrel structure and specific porins porins like LamB and ScrYshow ScrY show an 18-stranded barrel with surface exposed loops. High degree of sequence homology among the porins porins of enter enterobact obacteria eria allowed allowed a homology homology based model built built for Salmonella typhi OmpC (357 aa without the signal peptide and 39 kDa Mol.Wt.  /monom  /mo nomer) er) (18). (18). Earlie Earlierr we have predi predicte ctedd the sequen sequentia tiall B-cell B-cell epitop epitopic ic region regionss of S. typhi OmpC using sequence analysis (19). Subsequently we have crystallised OmpC (20) and the crystals diffract to low resolution. The homology based model will be used in the structure determination of OmpC using Molecular Replacement (MR) method. In the absence of crystal structure, the model is found useful to interpret the results obtained frommapping chemicalofmodification andwith immunochemical studies, towards conformational epitope S. typhi OmpC Salmonella and enterobacterial porin specific monoclonal antibodies. The functional implications of the model are discussed in this study.  Materials and Methods

Model building and energy minimization were carried out on a Silicon Graphics Iris Crimson workstation using Biosym InsightII 95.0/Homology and Discover modules (Biosym Technologies, San Diego). The energy calculations were done initially with the consistent valence force field (CVFF) and later on with AMBER force fields respectively (21). Electrostatic and solvent accessibility calculations were done with DELPHI and SOLVATION modules of InsightII. Trimer generation and final refinements were done using XPLOR (22). Molscript (23) was used to generate the figures. WGCG package (24) was used for sequence analysis and JOY (25) was used to represent the multiple sequence alignment with structural details. Sequences used in this study study were taken from EMBL and SwissProt and the referreference structures were from PDB (1OMF, 1PHO). Structure Based Sequence Alignment 

Structural superposition of reference structures E. coli OmpF and PhoE identified the structurally conserved regions (SCRs) among them. The multiple sequence alignment of S. typhi OmpC and E. coli OmpF & PhoE identified 9 homologous regions with gaps inserted at SCRs between OmpF and PhoE. Final alignment was made with the help of secondary structure information of OmpF and PhoE. Manual modification of the multiple sequence alignment was done to make sure there was no inserted gaps in the SCRs. Seven variable regions were found in the overall alignment.  Modelling of Structurally Conserved Regions

Coordinates for SCRs of OmpC were assigned from corresponding SCRs of OmpF and PhoE. Though OmpF and PhoE share structural homology in these regions, to assign the coordinates for OmpC, the respective SCRs of reference proteins were selected based on the sequence identity. The residues that differed in OmpC with respect to the reference structure were labeled as mutated residues for the model refinement. In case the percentage identity for a SCR of OmpC was same with both reference SCRs, selection was based on the origin of preceding and following SCRs of that particular conserved region of OmpC. In the case of SCR, SC R, corresponding to the β-strand 16 of OmpC, though it matches better with PhoE, coordinates were assigned from OmpF since the structure for strand 1 was taken from OmpF and strands 1 and 16 form a salt bridge bridge giving a pseudo cyclic structure. structure. Reference structures were superimposed before transferring the coordinates to OmpC model, to ensure the alignment of structural segments at the junctions.  Modeling of Variable Regions

The regions in between two SCRs were considered as structurally variable regions (V1-V8) and they show significant difference in terms of sequence as noticed from

 

the multiple sequence alignment. The variable regions, except for loop 3, are surface exposed (loops) in OmpF and PhoE. To To model the variable regions in OmpC, OmpC, loop search was done using high-resolution structures structures (>2Å) in PDB. Best-matched loops were selected based on the the following criteria: a) Loops with similar topolotopology and orientation b) Loops with low RMSD at the flanking regions. These variable segments were modelled using the backbone structure of the best-matched loop from the PDB, which resulted in acceptable topology and orientation. Part of  the structure was taken from reference structures for some variable regions. Thus all the variable regions could be modelled.  Refinement of the Initial Monomer Model

The refinement of the structure was essentially done in different levels to relieve the short contacts and bad geometry geometry.. Side chains of mutated residues were assigned from the rotamer library. Constrained energy minimization was done at the junction point between two segments if in case the two segments were taken from OmpF and PhoE. Side chains of mutated residues in SCR and all atoms in loop residues were relaxed using a few cycles of energy minimization. The resulting initial model was energy minimised initially using CVFF and then with AMBER force fields with steepest descent and conjugate gradient minimisers. Trimer Generation and Model Assessment 

Since the porins are present as trimers in vivo, any conformational analysis of porin will be more meaningful only if it is based on the trimer context. The S. typhi OmpC trimer was generated using the symmetry information of  E. coli OmpF. Trimer model was energy minimised using XPLOR to relieve short contacts, which might have been introduced while trimer generation. Stereochemistry of the model was analysed using PROCHECK (26). The coordinates of the model have been deposited in PDB (1IIV).  Electrostatic and Surface Accessibility Calculation Cal culation

Electrostatic potential was calculated using the DELPHI program. The linear Poisson-Boltzmann equation was solved, using Amber fractional charges for each protein atom. Dielectric constant of 2 and 80 were used for inside and outside the protein respectively. Program SOLVATION was used to calculate the surface accessibility with the probe radius of 1.4Å. Surface accessibility of both monomers and trimers were calculated and compared. Prediction of Sequential Epitopes

Sequential epitopes of S. typhi OmpC predicted earlier, based on sequence alignment, has been modified using the 3-D model. The modified ranking strategy is based on the antigenic index (AI) (27), ratio of variability in terms of sequence [Rv (seq) = no of non-conserved residues in loop region based on multiple sequence alignment /total number of residues in loop region] and the ratio of variability in terms of structure [Rv (str) = no of non-conserved residues in loop region based on the structural superposition /total number of residues in loop region] and surface accessibility (SA) in the context of the monomer and trimer.  Results and Discussion

The homology based model was built to find the structurally unique regions of

S.

typhi, which are likely to be exposed outside of the outer membrane and to outline

the features of its antigenic loops. Comparison of OmpC model with OmpF and PhoE crystal structures shows overall structural conservation in the invariant, membrane spanning, regions and significant differences in the loop regions.

263  Homology Model of Surface Antigen OmpC

 

264  Arockiasamy and Krishnaswamy 

 Model Building and Validity Validity of the Model

Porins from enterobacteria show high-level sequence sequence similarity though there is less homology observed between them and other bacteria (Table I). The level of  sequence similarity is reflected in the structure also (28). All non-specific porins seem to have a 16-stranded β-barrel structure. Since S. typhi OmpC showed more homology with E. coli OmpF and PhoE, these crystal structures were taken as reference to build the OmpC model. Though  Rhodopseudomonas capsulatus and  Rhodopseudomonas blastica porin structures are known, they were not included in reference structures due to poor sequence homology with OmpC. Table I

The percentage identity (I) and similarity (S) of S. typhi OmpC, E. coli OmpF, E. coli PhoE,  R. capsulatus and R. blastica porin sequences are given.

S. typhi   I OmpC S  E. coli   I OmpF S  E. coli   I PhoE S  R. capulatus   I porin S

 E. coli OmpF 63 77 -

E. coli PhoE 64 78 65 80 -

R. capulatus porin 22 48 19 48 18 49 -

R. blastica porin 22 51 22 49 23 48 28 55

OmpF and PhoE structures were compared and 8 structurally conserved regions, 4 variable regions and a single residue insertion in variable region 3 (loop 3)

 E. coli

were found. These two structures were superimposed with RMSD of 0.76Å over 1244 backbone atom pairs. The SCR regions cover 92.8% of the total residues and show 79% similarity in the sequence. SCRs of OmpF and PhoE comprise all membrane spanning and periplasmic facing and part of some of the exposed loop regions. Alignment of OmpC, OmpF and PhoE picked up 8 homologous regions, at the sequence level, and helped locating the corresponding SCRs in OmpC. The SCRs include identical positions and conservatively replaced positions of amino acids in membrane spanning β-strands and part of other regions. The solvent accessible regions of the β strands represent the membrane buried segments (Figure 1). The loop regions are exposed outside of the cell. S. typhi OmpC has some of the largest loops. Loops 6,7 and 8 show some structurally defined regions. Moreover the number of stabilising side chain and main chain hydrogen bonds are more for the larger loops of S. typhi OmpC. This probably reflects the minimisation of energy done in the absence of interacting molecules such as receptors and probably reflects the stabilisation required. The SCR regions were modelled first and then followed by variable regions. 11 regions on S. typhi OmpC comprising all transmembrane strands were assigned coordinates from OmpF and PhoE. The variable regions V1, V2, V4, V5 and V6 were built based on loop searches. Since there was no good match found for V3 using loop search the structure was subsequently assigned from OmpF based on the sequence match (71% similarity and 56% identity). V3 was split in to two segments before the structure was assigned, as there was a gap due to insertion of Serine 121 of PhoE in that SCR. Since part of the variable regions, at both ends of V7 and V8, of OmpC showed good match with SCRs of OmpF and PhoE, coordinates were assigned from PhoE on the basis of higher similarity and the core regions of these loops were assigned from loop search. The residues (183-184) forming turn-5 were modelled as SCR by taking the backbone from PhoE. Energy minimization with monomer and generated trimer resulted in the final model, which was checked using PROCHECK and the present model (Figure 2a, 2b) represents a valid structure (PDB code: 1IIV).  Model Analysis

OmpC folds, as expected from the sequence homology, as  E. coli OmpF and PhoE in the transmembrane and periplasmic facing regions. The OmpC trimer S. typhi

 

model was superimposed with OmpF and PhoE trimers at SCR regions with the RMSD of 1.21 and 1.37Å respectively, over 2544 backbone atom pairs. Superimposed monomers are seen in Figure 3. Variable regions of OmpC, comprising all surface exposed loops, are unique in terms of structure. Core regions of  the membrane spanning b-strands of  R. capsulatus and  R. blastica could be reasonably superimposed with OmpC, OmpF and PhoE (with the RMSD ranging from 1.40 to 1.95Å over 492 backbone atom pairs in monomer level), in spite of the low sequence homology. homology. The variations in the surface exposed regions could be due to evolutionary insertions and deletions which would have helped different bacteria to adjust to their varying environmental conditions and the interaction with their

265  Homology Model of Surface Antigen OmpC

 R. capsulatus hosts, in porins case ofhave animal and human For short example and  R. blactica simple barrel pathogens. structure with exposed loops unlike in Gram-negative bacteria where loops are long and are more variable. Variable regions V4 to V8 in OmpC have more number of amino acids than OmpF and PhoE indicating that S. typhi OmpC has unique variable surface exposed regions.  Electrostatic potential

The electrostatic potential surface of E. coli OmpF, PhoE and S. typhi OmpC porins show different spectrum of charge distribution at the cell surface (Figure 4a). The difference in electrostatic potential at the exposed regions, which might involve in antigen-antibody,, host-parasite interaction, phage attachment and the solute preferantigen-antibody ence at the pore entrance, could play a key role in terms of their function, specificity and antigenic variation. These surface exposed regions can be engineered to show different spectrum of charges at the cell surface, to understand the role of  charged amino acids in bringing about the host-bacterial interaction and their role in infection and host immunity. Electrostatic properties of porins can also explain and distinguish the role of variable regions in terms of physiological relevance other than immunological importance. Electrostatic properties of porins provide possible structure based explanation for their biological specificity towards anions or cations as noted earlier (29,30). This specificity is conferred by the charge distribution at the pore entrance and several mutational and modelling studies on loop 3 indicate the importance of the charged amino acids at the pore constriction zone (31). The counterparts of S. typhi OmpC in  E. coli and S. typhimurium have been shown to be cation selective. The electrostatic 2a

2b

Figure 2 a & b:The

Molscript ribbon diagram of the monomer (Figure 2a: View along the membrane) and trimer (Figure 2b: View down the pore) of S. typhi OmpC. The loops L1 to L8 are marked in the monomer. The loops are exposed outside of the cell and the short turns, at the bottom of the β-barrel, face the periplasmic space. M1, M2 and M3 M3 represent the monomers. monomers.

 

266  Arockiasamy and Krishnaswamy 

potential calculation shows that S. typhi OmpC could also be cation selective due to enriched negative charges at the pore entrance (Figure 4b). While going down the bottom of the pore, one could see only marginal differences between S. typhi OmpC and the reference porins in the charge spectrum as observed in the earlier studies. Surface Accessibility

Comparison of accessibility of the surface exposed variable regions of OmpC, OmpF and PhoE resulted in interesting observation when they were analysed in monomer and trimer levels. Though V1, V2 and V4 have more accessible area in monomer there is a regions reasonable context of interface. the trimerV2 (Table II) due to the level, fact that these are decrease involved in in the inter subunit of one subunit comes in contact with the neighbouring subunit and there is a latching by V2, which helps in trimer stability (32). There are not much variation in the amount of surface exposed area among V5, V6, V7 and V8 since they are away from trimer interface and are not involved in inter-subunit interaction. The results of comparison helped identify more exposed regions that could be of interest to design further experiments and also to provide structure based explanation for the experimental results got elsewhere as discussed below.  Model Implications

Porins have been shown to be potential surface antigens to elicit protective immune response in animal models. The homology model provides a structural basis of the antigenic variation, of exposed loops, which make the porins distinguishable in terms of their varying antigenic properties. The three dimensional model also helps explain properties of the conformational epitopes and uniqueness in terms of antigenecity. The folding of buried or TM regions are similar in enterobac enterobacteria, teria, while the exposed loops show the difference. This could explain how monoclonal antibodies raised for porins of Salmonella, which recognise the porin trimer on the cell surface (33), are able to distinguish between E. coli and Salmonella porins. This shows the existence of specific structural features in the overall exposed regions in the context of the trimer. Since the native trimers were used for immunization and also some MAbs recognise only the trimer and not the denatured monomers, it is obvious that the exposed surface area display differential specificity, which results in cross reaction and uniqueness. The MAbs have been shown to be useful in passive therapy against Salmonella infection and also demonstrated to be useful in early diagnosis of  typhoid fever (34,35). In the absence of complex crystal structures of Porin-MAbs, this model has been found useful to explain the experimental results obtained using chemically modified derivatives of S. typhi OmpC and radio labelled antibodies to map the conformational epitopes, specific to Salmonella porins and a common epitope shared by enterobacterial porins, on the surface exposed region (36). Synthetic peptides corresponding to V6 and V7 were shown to induce protective immunity (37). The success of these could also lie in the the better presentation afforded afforded by these loops which show greater stabilisation stabilisation in terms of hydrogen bonding (Figure 1). 1). The possibility of synthetic V4, V5 and V8 inducing such response could be tried out since V4 and V5 are better exposed than other variable regions. Combination of these regions would be more useful in such studies where one can avoid preparing the whole protein from pathogenic bacteria. Puente et. al., (6) have shown that S. typhi OmpC can be used as a carrier for expressing heterologous epitopes on the cell surface. By altering the loops V4 and V6, to study the expression of 18 amino acid long rotaviral epitope insert, it was shown that expression in V6 (loop 6) was better than in V4 (loop 4). This could be explained model, as V4 is better exposed thanV4V6ofinOmpC the structure since the exposure ofusing V6 isthe limited by the neighbouring loops (38). has 8 extra amino acids than PhoE and OmpF and also more variable. The possibility of V5, V7 and V8 being useful in such a study is more valid in the structural point of view. This study indi-

 

267  Homology Model of Surface Antigen OmpC

Figure 1:

final structure based sequence alignment of theThe S. typhi OmpC, E. coli OmpF and E. coli PhoE are shown. The sequences are only of of the mature peptide. The SCRs are marked with the continuous line over the corresponding regions. The residues forming beta strand are marked in blue, alpha helix forming residues are in red and the 3 10 helix formers in maroon. The solvent inaccessible residues are given in upper case. The solvent accessible residues are given in lower case. The residues forming hydrogen bond to the mainchain amide are in bold and those forming hydrogen bond to mainchain carbonyl are underlined. The loops (V1-V8) are indicated above the corresponding regions.

Figure 3 a & b:

The structural superposition of the

typhi OmpC (red),  E. coli OmpF monomers of S. (green) and  E. coli PhoE (blue). The figure was created using InsightII of the BIOSYM software. (a) View along the membrane, (b) View from the periplasmic space.

 

268  Arockiasamy and Krishnaswamy 

4a 4b

Figure 4 a & b:

Comparison of the electrostatic potentials of porin trimers S. typhi OmpC (lower left panel),  E. c oli OmpF (upper left panel) and  E. c oli PhoE (upper right panel). The potentials were calculated and displayed using DELPHI of BIOSYM software. The negative electrostatic potential regions are in red and the positive regions are in blue. The region, from where the slice of the electrostatic potential is taken, is shown in of thethe lower right panel withsurface reference to the Cα trace S. typhi OmpC trimer. The variations in electrostatic charge distribution a) towards the top of the trimers and b) near the membrane interface regions are shown.

 

cates the chances of S. typhi OmpC serving as a potential surface antigen to carry epitopes from other pathogens to develop diagnostic kits and its possible use in multivalent vaccine design. Structural constraints like packing of the loops may play a key role in accommodating the extra sequences of different length, in the variable regions. Moreover the loops are not exposed vertical to the plane of the membrane but are packed towards the axis passing through the pore, making some of the loops more accessible than others (Table II). The capacity of these regions to accommodate foreign sequences, with varying length, needs to be tested out.

269  Homology Model of Surface Antigen OmpC

Table II

The ranking of the antigenic loops on the basis antigenic index (AI), ratio of variability in terms of sequence [Rv (seq) = no of non-conserved residues loop based on multipleresidues sequenceinalignment (n2)/total of structural residues inalignment loop region] ratio of variability in terms of  structure in [Rv (str)region = no of non-conserved loop region based onnumber multiple (n2*)and /total number of residues in loop region] and surface accessibility (SA) per atom in the context of the monomer (SAM) and trimer(SAT). Lo Loo op

V1 (21-33) V2 (60-75) V4 (151-172) V5 (194-218) V6 (237-264) V7 (288-310) V8 (327-350)

Anti Antig genec enecit ity y Index

AI 1.29 0.93 1.25 0.96 1.00 0.82 0.70

No. of  non-conserved residues (seq) n2 3 9 12 15 16 12 10

AI/Rv (seq)

0.30 0.52 0.68 0.57 0.57 0.43 0.29

No. of  non-conserved residues (str) n2* 11 14 21 21 24 18 13

Surface AI/Rv (seq) Rank  Surface accessibility AI/Rv (seq)  /Atom X accessibility X (monomer) SAM /Rv (str)  /Atom SAT (A2) SAM (A2) (trimer) SAT (A2) 5.11 1.30 3.57 0.91 VI 4.85 2.21 2.16 0.98 V 4.50 2.92 3.60 2.33 I 4.07 1.95 4.21 2.02 II 2.40 1.17 2.38 1.16 III 3.16 1.06 3.18 1.07 IV 4.13 0.65 4.04 0.63 V VIII

Since porins were found to be B-Cell mitogens, attention have been focused on defined regions involved in this particular activity (39). Results of these reports indicate that synthetic peptides corresponding to the exposed regions could be more useful to delineate the residues involved in T-Cell independent B-cell induction and T-cell proliferation itself. Since defined fragments of  E. coli OmpF (153F:174V) and (157Y:174V) have been shown to activate macrophages (39), the corresponding regions identified from the structural alignment in S. typhi OmpC (145F:174Y) are likely to be involved in macrophage activation. This might explain the involvement of OmpC in infection and pathogenesis since OmpC is expressed throughout the infection period. Moreover the presence of antiporin antibodies in human typhoid sera makes it a promising candidate antigen to be explored. Lakshmi Mundkur (34) has shown that purified porin from S. typhi could induce T-Cells in vitro conditions which again shows the importance of S. typhi porin in cell mediated immunity. This has been supported by the results that Salmonella porin could induce lymphocytes to secrete cytokines (5). The need for the 3D structure was pointed out in the study of mapping of antigenic determinants of OmpC and OmpD from S. typhimurium (40,41) for accurate interpretation of the MAb reactivity. reactivity. We had earlier predicted the sequential epitopes of  S. typhi OmpC (19) based on the multiple sequence alignment, crystal structures of   E. coli OmpF and PhoE and partial modelling of two of the variable regions V1 and V4. In the present study, variable regions have been taken along with 3 flanking residues at both the ends in order to provide a presentation framework for the epitopes. These predicted regions were modified based on the surface accessibility of  the loop regions in the context of the monomer and the trimer from OmpC model. For example V2 is better exposed in the monomer model. However it is less exposed in the context of the trimer as it is involved in inter subunit locking. V1, being the shortest loop, is not well exposed in the trimer and therefore the possibility of being recognised by MAbs in the trimer context could be low (Table II). V3 is not included since it forms theV6 pore constriction in the middle of the barrel andinisthis not analysis surface exposed. Though forms the longest loop, the end of the loop goes down towards pore unlike in  E. coli OmpF.

 

270  Arockiasamy and Krishnaswamy 

The ranking of the possible B-cell epitopes (Table II) provides a rational strategy for choosing between the epitopes. The chances of a particular stretch stretch of a surface exposed region being the best candidate for experimental applications is tested taking into account the antigenic index, variability in terms of sequence alignment, the surface accessibility of the loops in the monomer context, variability based on the structural alignment and the surface accessibility of the loop residues in the context of the trimer. Any particular region, which ranks high based on these conditions, is considered to be a possible candidate for experimental design and the ranking strategy helps selecting one particular region over the other. The present study suggests that the best epitopes are V4, V5, V6 and V7 and the less likely ones are V8, V2 and V1, based on the trimer model. The superposition of three enterobacterial porins: S. typhi OmpC (model),  E. coli OmpF & PhoE and two other from non-pathogenic origins:  R. capsulatus and  R. blastica porin, have helped to make structure based multiple sequence alignment. This has led us to come up with sequence profiles from core regions of all 16 membrane spanning strands, which fall in the SCRs, to predict transmembrane β-stranded proteins from the sequence (42).  Acknowledgement

We are thankful to Profs. VR. Muthukkaruppan and K. Dharmalingam for encouragement and help. National Facility for High Resolution Graphics at Bioinformatics centre, Madurai Kamaraj University, supported by DBT, Govt. of  India is gratefully acknowledged. A. A is a recipient of a fellowship (NET) from CSIR. The research grant (SP/SO/D32/97) from DST, Govt. of India, is duly acknowledged.  References and Footnotes

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 Date Received: June 19, 2000

Communicated by the Editor Dino Moras 

271 Homology Model of Surface Antigen OmpC