The impact of length variations in the L2 loop on the structure and thermal stability of non-specific porins: The case of OmpCs from the Yersinia pseudotuberculosis complex

Porins are integral proteins of the outer membranes of gram-negative bacteria. In membranes, they exist as homotrimers and the L2 loops contribute to their stability. Comparison of OmpC porins of the Yersinia pseudotuberculosis complex with other enterobacterial porins demonstrated L2 loop length diversity, which is caused by varying numbers of dipeptide/tripeptide repeats. The OmpC porins are highly homologous to each other, and they can be subdivided into five isoforms based on their L2 loop structure. Optical spectroscopy and SDS-PAGE experiments revealed that particularities of the L2 loops affected the structure and thermal stability of the porins. Thermal denaturation studies showed that porins with shorter loops, compared to porins with longer loops, had more stable tertiary and less stable secondary and quaternary structures. According to our comparative modeling results, the L2 loops differ in their structure by adopting different spatial positions and forming different polar bonds with a neighbor monomer. The replacement of asparagine with arginine at the C-terminus of the L2 loop shifts the loop upwards and causes the loss of contacts with the arginine clusters within the pores. The increase in the length of these loops ensures that they shift down toward the pore and restore their contacts with arginines on the channel wall, as is the case in classical nonspecific porins. Despite the fact that the surface charge density varies considerably among the OmpC porins, the L2 loops form a typical negatively charged region in the center of the trimer.     

Homology models of the Yersinia pseudotuberculosis and Yersinia pestis general porins and comparative analysis of their functional and antigenic regions

The amino acid sequences of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) have recently deduced but their three-dimensional structures were not known. These sequences were analyzed using the servers 3D-PSSM and PredPort. The YPS and YPT porins were shown to have a high degree of identity (above 50%) in primary and secondary structures. The three-dimensional models of the Yersinia pseudotuberculosis porin (YPS) and Y. pestis porin (YPT) were obtained using the homology modeling approach, SWISS-MODEL Protein Modeling Server and 3-D structure of PhoE porin from E. coli as template. The superposition of the Calpha-atoms of the monomers of the Yersinia porins and PhoE porin gave a root mean square deviations of 0.47 A and 0.43 A for YPS and YPT respectively. Yersinia porins were found to be very similar in their three-dimensional structure to other non-specific enterobacterial porins, having the same features of overall fold and disposition of loop L3. The intrinsic structures of the monomer pores of YPS and YPT were investigated and their conductances were predicted with the program HOLE. The good correspondence between the theoretical and experimental magnitudes of YPS conductance was found. The Yersinia porins were determined to be unusual in containing the substitution, Glu replaced by Val, in a highly conserved pentapeptide (Pro-Glu-Phe-Gly-Gly-Asp), located in the loop L3 tip that disturbs the functionally important cluster of the acidic amino acids in the constriction site. Comparative analysis of structural organization of YPS and E. coli OmpF porin in the regions involved in subunit association and pore lumen was performed. The YPS porin functional properties were predicted. The differences between these porins in polar interactions playing a significant role in stabilization of the porin trimers were found and discussed in term of the variations in trimer stability. The Yersinia porins were shown to have the highest degree of the structural similarity. The differences between the porins were observed in their external loops. Their loops L6 and loops L8 showed 71.4 and 52.9% of sequence identity, respectively. The arrangement of charged residues clustered in the channel external vestibule of these porins was found to be also different suggesting the possible differences in their functional properties. The surface exposed regions of Yersinia porins involved in their potential sequential antigenic determinants were compared. The structural basis of their cross reactivity and antigenic differences is discussed.     

Prediction of B-cell epitopes forSalmonella typhi OmpC

The possible B-cell epitopes of the outer membrane porin OmpC ofSalmonella typhi have been identified, using the primary structure of the protein, by means of multiple sequence alignment and the known molecular structure of two other porins. From the analysis, 8 regions were identified as immunodominant and these were ranked based on antigenic index and the ratio of the number of nonconserved residues to the fragment length. Model building of the top two ranked regions show the tendency to form loop structures supporting the possibility of these being candidate epitopes.     

A comparative analysis of the spatial structure of nonspecific porins from <Emphasis Type="Italic">Yersinia ruckeri</Emphasis> using optical spectroscopy and molecular modeling

The spatial organization of outer-membrane porins is studied by optical spectroscopy and molecular modeling. It was found that the OmpF and OmpC porins from Yеrsiniа ruckeri are β-structured membrane proteins typical of the pore-forming proteins of other Gram-negative bacteria. The spatial structures of monomers and trimers of the OmpC and OmpF porins from Y. ruckeri are simulated using methods of structural bioinformatics. It was found that the structural stability of the more thermostable OmpF trimer is sustained by a greater number of hydrogen bonds and hydrophobic interactions. The main differences of the spatial structures of the test porins are observed in the structure of their outer loops. There are three tryptophan residues in the molecules of the OmpC and OmpF porins of Y. ruckeri. It is demonstrated by moleculardynamics methods that after thermal denaturation the solvent accessibility of the Trp212 residue in OmpF porin increased by two times, while the solvent accessibility of a Trp184 residue in OmpC porin was not increased. It is hypothesized that the red-shifted tryptophan fluorescence spectrum of OmpF porin during thermal denaturation is due to the behavior of the Trp212 residue.     

A sequence-specific DNA-binding protein interacts with the xlnC upstream region of Streptomyces sp. strain EC3

Abstract With the recent resolution of the crystal structures of several bacterial porins, it is worthwhile to define the generality of their organization throughout the Enterobacteriaceae. The distribution of specific epitopes was analysed among various Gram-negative bacterial porins using anti-peptide antibodies specific to exposed, transmembrane spanning, or pore-forming regions of Escherichia coli porins. Anti-peptide antibodies which recognized the exposed epitopes indicated a great variability among the bacterial porins analysed. Interestingly, an antigenic site located in the internal loop L3 constricting the pore diameter was present in the majority of the bacterial porins tested. Two epitopes located in domains involved in subunit interaction were also highly conserved. The presence of these common peptides suggested a conservation of specific regions involved in the functional organization of the enterobacterial porins.     

Porins OmpC and PhoE of Escherichia coli as specific cell-surface targets of human lactoferrin. Binding characteristics and biological effects.

The binding of lactoferrin, an iron-binding glycoprotein found in secretions and leukocytes, to the outer membrane of Gram-negative bacteria is a prerequisite to exert its bactericidal activity. It was proposed that porins, in addition to lipopolysaccharides, are responsible for this binding. We studied the interactions of human lactoferrin with the three major porins of Escherichia coli OmpC, OmpF, and PhoE. Binding experiments were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds to the purified native OmpC or PhoE trimer with molar ratios of 1.9 +/- 0.4 and 1.8 +/- 0.3 and Kd values of 39 +/- 18 and 103 +/- 15 nM, respectively, but not to OmpF. Furthermore, preferential binding of lactoferrin was observed on strains that express either OmpC or PhoE. It was also demonstrated that residues 1-5, 28-34, and 39-42 of lactoferrin interact with porins. Based on sequence comparisons, the involvement of lactoferrin amino acid residues and porin loops in the interactions is discussed. The relationships between binding and antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological studies revealed that lactoferrin can act as a blocking agent for OmpC but not for PhoE or OmpF. However, a total inhibition of the growth was only observed for the PhoE-expressing strain (minimal inhibitory concentration of lactoferrin was 2.4 mg/ml). These data support the proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the stability and/or the permeability of the bacterial outer membrane.     

Molecular modelling of epitope presentation using membrane protein OmpC

Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops (fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both the chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop, in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.     

Immunological approach of assembly and topology of OmpF, an outer membrane protein of Escherichia coli.

Various monoclonal antibodies (MoF) directed against cell-surface-exposed epitopes of OmpF, one major outer membrane pore protein of Escherichia coli B and K-12, have been used to study the assembly and the topology of the protein. This paper firstly describes the characterization of the OmpF epitopes recognized by the various monoclonal antibodies. A comparison between OmpC, OmpF and PhoE porins with respect to their primary amino acid sequence and their cell-surface exposed regions allows us to propose a rough model including 2 antigenic sites. The second part is focused on the assembly of the OmpF protein in the outer membrane. Various forms, precursor, unassembled monomer, metastable oligomer (pre-trimer) and trimer are detected with immunological probes directed against OmpF during a kinetic analysis of the process. The requirement for a concomitant lipid synthesis during the trimerization has been demonstrated by investigating the presence of a specific native epitope. The role of lipopolysaccharide during the stabilization of the conformation is discussed with regard to the various steps of assembly.     

OmpC-like porin from outer membrane of Yersinia enterocolitica: Molecular structure and functional activity

OmpC-like porin was isolated from the outer membrane (OM) of Yersinia enterocolitica cultured at 37°C (the “warm” variant) and its physicochemical and functional properties were studied. The amino acid sequence of OmpC porin was established, and the primary structure and transmembrane topology of this protein were analyzed in comparison with the OmpF porin isolated from Y. enterocolitica cultured at 6°C (the “cold” variant). Both porins of Y. enterocolitica had a high homology degree (65%) between themselves and with OmpC and OmpF porins from OM of Escherichia coli (58 and 76% homology, respectively). The secondary structure of OmpC and OmpF porins from OM of Y. enterocolitica consists of 16 β-strands connected by short “periplasmic” and longer “extracellular” loops with disordered structure, according to the topological model developed for porins of E. coli. The molecular structures of OmpC and OmpF porins of Y. enterocolitica have significant differences in the structure of the “extracellular” loops and in the position of one of three tryptophan residues. Using the bilayer lipid membrane (BLM) technique, pores formed by OmpC porin of Y. enterocolitica were shown to differ in electrophysiological characteristics from channels of OmpF protein of this microorganism. The isolated OmpC porin reconstructed into BLM displayed functional plasticity similarly to OmpF protein and nonspecific porins of other enterobacteria. The conductivity level of the channels formed by this protein in the BLM was regulated by value of the applied potential.     

OmpC-like porin from Yersinia pseudotuberculosis: Molecular characteristics, physico-chemical and functional properties

Pore-forming protein from the outer membrane of Yersinia pseudotuberculosis cultured at 37°C has been isolated and characterized. Comparative analysis of the primary and three-dimensional structures of this protein and of OmpC porin from E. coli was carried out, functional properties of these proteins have been studied using bilayer lipid membranes (BLM) technique. The degree of homology, molecular mass and pore-forming properties of the isolated porin was found to be closer to those of OmpC porin from E. coli than OmpF porin from Y. pseudotuberculosis. The value of the most probable conductivity of OmpC porin from Y. pseudotuberculosis (0.18 pS) in BLM corresponded to the conductivity of the native trimer of this protein. Using CD spectroscopy, the porins investigated were shown to belong to the β-structured proteins. Data of the primary structure and intrinsic protein fluorescence revealed essential differences in localization and microenvironment of tryptophan residues in the porins investigated. Participation of external loops L2 and L6 in the formation of the antigenic structure of OmpC porin from Y. pseudotuberculosis was demonstrated. On the basis of crystal structure of osmoporin from Klebsiella pneumoniae, three-dimensional models of the monomer and trimer of the Y. pseudotuberculosis porin were obtained. Using Web server AGGRESCAN, the localization of protein structure sites with the increased aggregation capability (hot spots) has been deter-mined. It turned out that some of these zones localize in the region of intramonomeric contacts in the porin trimer; however, a large part of them is located on the external surface of the β-barrel. The process of thermal denaturation has been studied and the melting points of the porins were determined. It was found that significant changes in the microenvironment of the indole fluorophores (especially tryptophan residues of spectral class I) took place in the process of the thermodenaturation of the proteins. These changes preceded the irreversible conformational transition observed for the E. coli porin at 77°C and for the Y. pseudotuberculosis porin at 70°C.     

Conformational analysis of the Campylobacter jejuni porin.

The major outer membrane protein (MOMP) of Campylobacter jejuni was purified to homogeneity by selective solubilization and fast protein liquid chromatography. The amino acid composition of the MOMP indicates the presence of cysteine residues. The amino-terminal sequence, determined over 31 residues, shows no significant homology with any other porin from gram-negative bacteria except in a discrete region. Immunocross-reactivity between Escherichia coli OmpC and the MOMP was analyzed, and a common antigenic site between these two porins was identified with an anti-peptide antibody. From circular dichroism and immunological investigations, the existence of a stable folded monomer, containing a high level of beta-sheet secondary structure, is evident. Conformational analyses show the presence of a native trimeric state generated by association of the three folded monomers; the stability of this trimer is reduced compared with that of E. coli porins. This study clearly reveals that the C. jejuni MOMP is related to the family of trimeric bacterial porins.     

Porins of Escherichia coli: unidirectional gating by pressure.

OmpC and PhoE porins of Escherichia coli were examined by the patch-clamp technique following reconstitution in liposomes, and were observed primarily in the open (conducting) state. With application of negative voltage and positive hydrostatic pressure, OmpC exhibited marked gating towards a more closed state whereas PhoE remained largely unaffected by pressure application. Hybrid chimeric OmpC-PhoE proteins showed an increased tendency for pressure-dependent gating as the OmpC proportion in the chimeric molecule increased. In addition, several PhoE mutants with amino acid substitutions and insertions in either the L3 or L4 loop of the monomer exhibited pressure sensitivity comparable with the wild-type OmpC porin. Our data support the structural plasticity model of porins and are consistent with the 'charge-screening-unscreening' hypothesis that describes how these proteins may exist in distinct conformations.     

A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has gained considerable attention for developing subunit vaccines against HIV-1 and also as a target to develop anti-HIV-1 drugs. These endeavors would be significantly enhanced by understanding the structural aspects of this loop. The structure of the full-length V3 loop has not been defined yet. Therefore, a novel modeling technique, termed `Fragment Fitting Approach′ (FFA), was developed to model the V3 loop. This technique utilizes fragments (³ 6 residue long) with local sequence and secondary structure similarity from unrelated proteins with known x-ray crystallographic structure and concatenating the fragments to build the model. A systematic search method was devised to identify the fragments using the combined criteria of sequence and secondary structure identity and/or similarity, predicted by a combination of methods. FFA requires partial three-dimensional coordinates of the target sequence to be modelled to get the overall coordinate path correct. The method was validated with nine disulfide-bonded loops from the Protein data bank. The modelled structures conform well with the corresponding x-ray crystallographic structures. As the models were built using the x-ray coordinates with reasonable resolution (£ 3 Å), they are expected to have stereochemically correct structures. The modelled V3 loop structure might assist in structure-based drug design of anti-HIV-1 agents targeted to this loop.     

A ′Fragment Fitting Approach′ to Model Disulfide Loops by Utilizing Homologous Peptide Fragments from Unrelated Proteins of Known Structures: Application to the V3 Loop of the HIV-1 Envelope Glycoprotein gp120

The V3 loop of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 has gained considerable attention for developing subunit vaccines against HIV-1 and also as a target to develop anti-HIV-1 drugs. These endeavors would be significantly enhanced by understanding the structural aspects of this loop. The structure of the full-length V3 loop has not been defined yet. Therefore, a novel modeling technique, termed `Fragment Fitting Approach′ (FFA), was developed to model the V3 loop. This technique utilizes fragments (³ 6 residue long) with local sequence and secondary structure similarity from unrelated proteins with known x-ray crystallographic structure and concatenating the fragments to build the model. A systematic search method was devised to identify the fragments using the combined criteria of sequence and secondary structure identity and/or similarity, predicted by a combination of methods. FFA requires partial three-dimensional coordinates of the target sequence to be modelled to get the overall coordinate path correct. The method was validated with nine disulfide-bonded loops from the Protein data bank. The modelled structures conform well with the corresponding x-ray crystallographic structures. As the models were built using the x-ray coordinates with reasonable resolution (£ 3 Å), they are expected to have stereochemically correct structures. The modelled V3 loop structure might assist in structure-based drug design of anti-HIV-1 agents targeted to this loop.     

Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.     

Protein loops on structurally similar scaffolds: database and conformational analysis

A general problem in comparative modeling and protein design is the conformational evaluation of loops with a certain sequence in specific environmental protein frameworks. Loops of different sequences and structures on similar scaffolds are common in the Protein Data Bank (PDB). In order to explore both structural and sequential diversity of them, a data base of loops connecting similar secondary structure fragments is constructed by searching the data base of families of structurally similar proteins and PDB. A total of 84 loop families having 2-13 residues are found among the well-determined structures of resolution better than 2.5 A. Eight alpha-alpha, 20 alpha-beta, 19 beta-alpha, and 37 beta-beta families are identified. Every family contains more than 5 loop motifs. In each family, no loops share same sequence and all the frameworks are well superimposed. Forty-three new loop classes are distinguished in the data base. The structural variability of loops in homologous proteins are examined and shown in 44 families. Motif families are characterized with geometric parameters and sequence patterns. The conformations of loops in each family are clustered into subfamilies using average linkage cluster analysis method. Information such as geometric properties, sequence profile, sequential and structural variability in loop, structural alignment parameters, sequence similarities, and clustering results are provided. Correlations between the conformation of loops and loop sequence, motif sequence, and global sequence of PDB chain are examined in order to find how loop structures depend on their sequences and how they are affected by the local and global environment. Strong correlations (R > 0.75) are only found in 24 families. The best R value is 0.98. The data base is available through the Internet.     

Sequence–structure relationships in RNA loops: establishing the basis for loop homology modeling

The specific function of RNA molecules frequently resides in their seemingly unstructured loop regions. We performed a systematic analysis of RNA loops extracted from experimentally determined three-dimensional structures of RNA molecules. A comprehensive loop-structure data set was created and organized into distinct clusters based on structural and sequence similarity. We detected clear evidence of the hallmark of homology present in the sequence–structure relationships in loops. Loops differing by <25% in sequence identity fold into very similar structures. Thus, our results support the application of homology modeling for RNA loop model building. We established a threshold that may guide the sequence divergence-based selection of template structures for RNA loop homology modeling. Of all possible sequences that are, under the assumption of isosteric relationships, theoretically compatible with actual sequences observed in RNA structures, only a small fraction is contained in the Rfam database of RNA sequences and classes implying that the actual RNA loop space may consist of a limited number of unique loop structures and conserved sequences. The loop-structure data sets are made available via an online database, RLooM. RLooM also offers functionalities for the modeling of RNA loop structures in support of RNA engineering and design efforts.     

Biological and immunological comparisons of Enterobacter cloacae and Escherichia coli porins

Abstract Bacteriocin susceptibilities indicate that during cloacin DF13 uptake the F porin of Enterobacter cloacae plays a similar role to that reported for the OmpF porin of Escherichia coli during colicin A entry. The translocatory activities of these two porins during the bacteriocin uptake can be substituted by the porins D and OmpC, respectively, under conditions not requiring the receptor binding step. Using anti-peptide antibodies, a peptide located in the internal loop L3 of the Escherichia coli OmpF porin was identified in the D and F porins of Enterobacter cloacae. The results demonstrated the existence of a close relationship between porins in terms of both antigenic determinants and bacteriocin susceptibilities.     

Characterization of Salmonella typhi OmpC and OmpF porins engineered with HIV‐gp41 epitope on the surface loops

Porins form trimers in the outer membrane and help transport nutrients and waste products across the bacterial cell membrane. Porin loops are suitable candidates as display systems due to their high immunogenicity and presentation at the bacterial cell surface. In this study, Salmonella typhi porins (OmpC and OmpF) engineered with the Kennedy peptide from gp41 of HIV were characterised. The chimeric OmpC carrying the Kennedy peptide in loop7 did not trimerise, whereas the chimeric OmpF with the epitope in loop5 formed trimers and also was recognised by the antibodies in the HIV patient serum. The results suggest that chimeric S. typhi OmpF may be taken further as a potential candidate to develop as an epitope display system. Proteins 2017; 85:657–664. © 2016 Wiley Periodicals, Inc.     

Crystal structure of osmoporin OmpC from E. coli at 2.0 A

Porins form transmembrane pores in the outer membrane of Gram-negative bacteria with matrix porin OmpF and osmoporin OmpC from Escherichia coli being differentially expressed depending on environmental conditions. The three-dimensional structure of OmpC has been determined to 2.0 A resolution by X-ray crystallography. As expected from the high sequence similarity, OmpC adopts the OmpF-like 16-stranded hollow beta-barrel fold with three beta-barrels associated to form a tight trimer. Unlike in OmpF, the extracellular loops form a continuous wall at the perimeter of the vestibule common to the three pores, due to a 14-residues insertion in loop L4. The pore constriction and the periplasmic outlet are very similar to OmpF with 74% of the pore lining residues being conserved. Overall, only few ionizable residues are exchanged at the pore lining. The OmpC structure suggests that not pore size, but electrostatic pore potential and particular atomic details of the pore linings are the critical parameters that physiologically distinguish OmpC from OmpF.