Analysis of structure-function relationships in Escherichia coli K12 outer membrane porins with the aid of ompC-phoE and phoE-ompC hybrid genes

Summary To study structure-function relationships in the outer membrane pore proteins OmpC and PhoE of Escherichia coli K12, we have constructed a series of phoE-ompC hybrid genes in which DNA encoding part of one protein is replaced by the homologous part of the other gene. The hybrid gene products were incorporated normally into the outer membrane, allowing their functional characterization. Combined with previous studies, the present results permit the identification of regions involved in determining functions and properties in which the native PhoE and OmpC proteins differ, such as pore characteristics, receptor activity for phages and binding of monoclonal antibodies. Most of these properties were found to be determined by multiple regions clearly separated in the primary structure. The combined phage and antibody binding data have demonstrated that at least five distinct regions in PhoE and OmpC are exposed at the cell surface. The locations of these regions are in agreement with a previously proposed model for porin topology.     

New pore protein produced in cells lysogenic for Escherichia coli phage HK253hrk

Outer membrane pore protein OmpC was identified as the receptor for the temperate Escherichia coli phage HK253hrk. The part of OmpC protein recognized by the phage was identified by using hybrid proteins in which parts of OmpC protein are replaced by the corresponding parts of the related PhoE protein. In contrast to other OmpC-specific phages, HK253hrk recognizes a part of OmpC within the C-terminal 50 amino acids of the protein. E. coli strains lysogenic for HK253hrk produce reduced amounts of OmpC protein, and produce a new pore protein instead. Expression of this new protein was temperature-dependent, i.e. low at 30 degrees C. The functioning of this new pore protein was characterized both in vivo by studying the uptake of beta-lactam antibodies and in vitro after reconstitution of the protein in black lipid films. Its effective pore size was larger than that of the OmpF pores of E. coli B. The new porin appears to be cation-selective. A comparison with the selectivity of the known OmpC and OmpF pores of E. coli showed that the new pore has a higher selectivity than OmpF but is less selective than OmpC. The new pore protein appears to function in E. coli K12 lysogens as the receptor for the phages HK187, HK189 and HK332.     

Role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein

To investigate the role of the cell surface-exposed regions of outer membrane protein PhoE of Escherichia coli K12 in the biogenesis of the protein, deletions were generated in two presumed cell surface-exposed regions of the protein. Intact cells expressing these mutant proteins were recognized by PhoE-specific monoclonal antibodies, which recognize conformational epitopes on the cell surface-exposed parts of the protein and/or were sensitive to a PhoE-specific phage. This shows that the polypeptides were normally incorporated into the outer membrane. When the deletions extended four amino acid residues into the seventh presumed membrane-spanning segment, the polypeptides accumulated in the periplasm. In conclusion, exposed regions of PhoE protein apparently do not play an essential role in outer membrane localization, which is consistent with the observation that these regions are hypervariable when PhoE is compared to the related proteins OmpF and OmpC. In contrast, the membrane-spanning segments are essential for the assembly process.     

Gene encoding a hybrid OmpF-PhoE pore protein in the outer membrane of Escherichia coli K12

Summary To study the structure-function relationship of outer membrane pore proteins of E. coli K12, a hybrid gene was constructed in which the DNA encoding amino acid residues 2–73 of the mature PhoE protein is replaced by the homologous part of the related ompF gene. The product of this gene is incorporated normally into the outer membrane. It was characterized with respect to its pore activity and its phage receptor and colicin receptor properties. It is concluded (i) that the preference of the PhoE protein pore for negatively charged solutes is partly determined by the amino terminal 73 amino acids, (ii) that part of the receptor site of PhoE protein for phage TC45 is located in this part of the protein, (iii) that colicin N uses OmpF protein as (part of) its receptor, (iv) that the specificity of OmpF protein as a colicin N receptor is completely located within the 80 amino terminal amino acid residues, whereas the specificity of this protein as a colicin A receptor is completely located within the 260 carboxy terminal amino acid residues, and (v) that the amino terminal 73 amino acid residues of PhoE protein span the membrane at least once.     

Insertion mutagenesis on a cell-surface-exposed region of outer membrane protein PhoE of Escherichia coli K-12

Amino acid residue arginine-158 of the outer membrane protein PhoE of Escherichia coli K-12 has been shown to be cell-surface-exposed [Korteland et al. (1985) Eur. J. Biochem. 152, 691-697]. To study the effects of small insertions in this region of the protein on its biogenesis and characteristics, a unique restriction site was created by site-directed mutagenesis in a plasmid carrying the phoE gene and oligonucleotides of 12-74 bp were inserted. The insertions did not interfere with incorporation into the outer membrane since (a) several monoclonal antibodies, directed against the cell-surface-exposed part of PhoE protein, bound to whole cells producing the altered proteins and (b) the proteins formed functional pores for the uptake of beta-lactam antibiotics. The binding of one monoclonal antibody and of the PhoE-specific phages TC45 and TC45hrN3 was disturbed by the insertions, showing that this region of the protein is immunogenic and is involved in the binding of both of these phages. The functioning of the mutant pores was characterized both in vivo by studying the uptake of beta-lactam antibiotics and in vitro after the reconstitution of the proteins in black lipid films. The pore characteristics changed depending on the nature of the inserted amino acids. Addition of a negatively charged amino acid resulted in decreased anion-selectivity, whereas insertion of a positive charge and deletion of a negative charge had only a small influence.     

Demonstration of a bacteriophage receptor site on the Escherichia coli K12 outer-membrane protein OmpC by the use of a protease

The Escherichia coli K12 outer-membrane proteins OmpA, OmpC, OmpF, PhoE, and LamB (all of transmembrane nature) can serve as phage receptors. We have shown previously that one OmpA-specific phage, Ox2, can give rise to the host range mutants Ox2h10 and Ox2h12, with the latter being derived from the former [Morona, R. & Henning, U. (1984) J. Bacteriol. 159, 579-582]. Unlike Ox2, both host range phages can use the OmpA and OmpC proteins as receptors and Ox2h12 is better adapted to the OmpC protein than Ox2h10. In a search for the site(s) of OmpC protein involved in phage recognition, it was found that proteinase K is able to cleave all of the proteins mentioned above. OmpC protein (Mr = 38306) could be cleaved from outside the cell by proteinase K resulting in two fragments of Mr approximately equal to 21000 and Mr approximately equal to 17500. The use of OmpC-PhoE hybrid proteins allowed us to assign the approximately equal to 21000-Mr fragment to the CO2H-terminal moiety of the protein. Proteinase K treatment of intact cells abolished their activity to neutralize the OmpC-specific phage Tulb and reduced this ability towards phage Ox2h12. The OmpA, OmpF, PhoE and LamB proteins were cleaved by the protease not in intact cells but only when acting on cell envelopes. The sizes of the OmpC protein fragments and the results obtained with the hybrid proteins very strongly suggest that the protein is cleaved from outside the cell at a region involving amino acid residues 150-178 of the 346-residue protein, which shows homology to two regions of the OmpA protein which are involved in its phage receptor site (loc. cit.). These areas also exhibit some homology to a region of the LamB protein which is thought to be part of this protein's receptor site [Charbit et al. (1984) J. Mol. Biol. 175, 395-401]. This suggests that there is a common denominator for proteinaceous phage receptor site because the LamB-specific phage lambda and phage Tulb are of completely different nature. We conclude that the region of the OmpC protein in question is cell-surface-exposed and acts as a phage receptor site.     

A comparative study on the genes for three porins of the Escherichia coli outer membrane. DNA sequence of the osmoregulated ompC gene

The DNA sequence of the ompC gene which encodes one of the outer membrane porins has been determined. The gene appears to encode a secretory precursor of OmpC protein consisting of a total of 367 amino acid residues with a signal peptide of 21 amino acid residues at its NH2-terminal end. The 5' end noncoding region including the promoter of the ompC gene is extremely [A-T]-rich, and the codon usage in the ompC gene is unusual as are those in genes for other abundant outer membrane proteins. The promoter sequence of the ompC gene was compared with that of the ompF gene, both of which are controlled by the osmoregulatory operon, ompB. The deduced amino acid sequence of the OmpC protein showed extensive homology with that of the other porins (OmpF and PhoE proteins). The homology in the primary amino acid sequences, as well as the coding DNA sequences among the porins, indicates that the structural genes for the three porins evolved from a common ancestral gene. Comparison of the amino acid sequences among the OmpC, OmpF, and PhoE porins will be discussed with regard to structure and function.     

Topology of outer membrane pore protein PhoE of Escherichia coli. Identification of cell surface-exposed amino acids with the aid of monoclonal antibodies

Monoclonal antibodies which recognize the cell surface-exposed part of outer membrane protein PhoE of Escherichia coli were used to select for antigenic mutants producing an altered PhoE protein. The selection procedure was based on the antibody-dependent bactericidal action of the complement system. Using two distinct PhoE-specific monoclonal antibodies, seven independent mutants with an altered PhoE protein were isolated. Among these seven mutants, five distinct binding patterns were observed with a panel of 10 monoclonal antibodies. DNA sequence analysis revealed the following substitutions in the 330-residue-long PhoE protein: Arg-201----His (three isolates), Arg-201----Cys, Gly-238----Ser, Gly-275----Ser and Gly-275----Asp. It is argued that amino acid residues 201, 238, and 275 are most likely directly involved in antibody binding and, therefore, exposed at the cell surface. Together with Arg-158, which was previously shown to be cell surface exposed as it is changed in phage TC45-resistant phoE mutants, these four positions show a remarkably regular spacing, being approximately 40 residues apart. A model is suggested in which the PhoE polypeptide repeatedly traverses the outer membrane in an antiparallel beta-pleated sheet structure, exposing eight areas to the outside which are all separated by approximately 40 residues.     

A comparative study on the phoE genes of three enterobacterial species. Implications for structure-function relationships in a pore-forming protein of the outer membrane

The cloned phoE genes from Enterobacter cloacae and Klebsiella pneumoniae are normally expressed and regulated in Escherichia coli K-12, and their products are correctly assembled into the outer membrane. Differences between the three PhoE proteins were found with binding of two out of ten monoclonal antibodies directed against the cell-surface-exposed part and in pore characteristics, but not in phage receptor function. The DNA sequences of the E. cloacae and K. pneumoniae phoE genes were determined and used to predict the primary structures of the encoded proteins. In the upstream non-coding regions, which showed more variations among the three genes than the coding regions, conserved sequences were identified which might be involved in regulation of phoE gene expression. Comparison of the predicted PhoE primary structures revealed a high degree of homology, with 81% of the amino acid residues being identical in all three proteins. Four small variable regions were found where differences are the most pronounced, corresponding to regions which were previously predicted to be exposed at the cell surface. Implications of the sequence comparison for structure-function relationships in PhoE protein are discussed.     

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.     

Construction of a series of ompC-ompF chimeric genes by in vivo homologous recombination in Escherichia coli and characterization of their translational products

Summary OmpC and OmpF are major outer membrane proteins and although they are homologous proteins, they function differently in several respects. As an approach to elucidate the submolecular structures that determine their differences, we have constructed a series of ompC-ompF chimeric genes by in vivo homologous recombination between these two genes, which are adjacent on a plasmid. The recombination sites in the chimeric genes were localized by means of restriction endonuclease analysis and nucleotide sequence determination. Most of the chimeric gene products were accumulated in the outer membrane. One of the chimeric gene products, with a fusion site in a central region between the OmpC and OmpF proteins, was normally expressed but not accumulated in the outer membrane. The trimeric structures of some of the chimeric gene products appeared to be extremely unstable in a SDS solution. From these results, domains contributing to the formation of specific structures in which the OmpC and OmpF proteins differ were identified. Bacterial cells possessing the chimeric gene products were also investigated as to their sensitivity to phages that require either OmpC or OmpF as a receptor component. With the aid of the chimeric gene products, the immunogenic determinants for three anti-OmpC monoclonal antibodies were found to be localized at different portions of the OmpC polypeptide: the N-terminal, central and C-terminal portions, respectively.     

PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate

This study was undertaken to investigate the proposed in vivo pore function of PhoE protein, an Escherichia coli K12 outer membrane protein induced by growth under phosphate limitation and to compare it with those of the constitutive pore proteins OmpF and OmpC. Appropriate mutant strains were constructed containing only one of the proteins PhoE, OmpF or OmpC, or none of these proteins at all. By measuring rates of nutrient uptake at low solute concentrations, the proposed pore function of PhoE protein was confirmed as the presence of the protein facilitates the diffusion of Pi through the outer membrane, such as a pore protein deficient strain behaves as a Km mutant. Comparison of the rates of permeation of Pi, glycerol 3-phosphate and glucose 6-phosphate through pores formed by PhoE, OmpF and OmpC proteins shows that PhoE protein is the most effective pore in facilitating the diffusion of Pi and phosphorus-containing compounds. The three types of pores were about equally effective in facilitating the permeation of glucose and arsenate. Possible reasons for the preference for Pi and Pi-containing solutes are discussed.     

Rise and fall of the delta globin gene

The complete nucleotide sequence of the gene phoE, which codes for the phosphate limitation inducible outer membrane pore protein of Escherichia coli K12 was established. The results show that PhoE protein is synthesized in a precursor form with a 21 amino acid residue amino-terminal extension. This peptide has the general characteristics of a signal sequence. The promoter region of phoE has no homlogy with the consensus sequence of E. coli promoter regions, but homologous sequences with the promoter region of phoA, the structural gene for alkaline phosphatase, were observed. The deduced amino acid sequence showed that the mature PhoE protein is composed of 330 amino acid residues with a calculated molecular weight of 36,782. A number of 81 charged amino acids was found scattered throughout the protein while no large stretches of hydrophobic amino acids were observed. Hydrophobicity and hydration profiles of PhoE protein showed five pronounced hydrophilic maxima which are all located in the region from the amino terminus to residue 212.When the deduced amino acid sequence of PhoE protein was compared with the established sequence of the OmpF pore protein, a number of 210 identical residues was found. Some aspects of the structure-function relationship of PhoE protein are discussed in view of the hydrophobicity and hydration profiles, and the homology between PhoE protein and OmpF protein.     

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.     

Identification of C/EBP basic region residues involved in DNA sequence recognition and half-site spacing preference.

C/EBP and GCN4 are basic region-leucine zipper (bZIP) DNA-binding proteins that recognize the dyad-symmetric sequences ATTGCGCAAT and ATGAGTCAT, respectively. The sequence specificities of these and other bZIP proteins are determined by their alpha-helical basic regions, which are related at the primary sequence level. To identify amino acids that are responsible for the different DNA sequence specificities of C/EBP and GCN4, two kinds of hybrid proteins were constructed: GCN4-C/EBP chimeras fused at various positions in the basic region and substitution mutants in which GCN4 basic region amino acids were replaced by the corresponding residues from C/EBP. On the basis of the DNA-binding characteristics of these hybrid proteins, three residues that contribute significantly to the differences in C/EBP and GCN4 binding specificity were defined. These residues are clustered along one face of the basic region alpha helix. Two of these specificity residues were not identified as DNA-contacting amino acids in a recently reported crystal structure of a GCN4-DNA complex, suggesting that the residues used by C/EBP and GCN4 to make base contacts are not identical. A random binding site selection procedure also was used to define the optimal recognition sequences for three of the GCN4-C/EBP fusion proteins. These experiments identify an element spanning the hinge region between the basic region and leucine zipper domains that dictates optimal half-site spacing (either directly abutted for C/EBP or overlapping by one base pair for GCN4) in high-affinity binding sites for these two proteins.     

PhoE protein pore of the outer membrane of Escherichia coli K12 is a particularly efficient channel for organic and inorganic phosphate

This study was undertaken to investigate the proposed in vivo pore function of PhoE protein, an Escherichia coli K12 outer membrane protein induced by growth under phosphate limitation, and to compare it with those of the constitutive pore proteins OmpF and OmpC. Appropriate mutant strains were constructed containing only one of the proteins PhoE, OmpF or OmpC, or none of these proteins at all. By measuring rates of nutrient uptake at low solute concentrations, the proposed pore function of PhoE protein was confirmed as the presence of the protein facilitates the diffusion of P_i through the outer membrane, such that a pore protein deficient strain behaves as a K_m mutant. Comparison of the rates of permeation of P_i, glycerol 3-phosphate and glucose 6-phosphate through pores formed by PhoE, OmpF and OmpC proteins shows that PhoE protein is the most effective pore in facilitating the diffusion of P_i and phosphorus-containing compounds. The three types of pores were about equally effective in facilitating the permeation of glucose and arsenate. Possible reasons for the preference for P_i and P_i-containing solutes are discussed.     

Characterization of the DNA binding and structural properties of the BRCT region of human replication factor C p140 subunit

BRCT domains, present in a large number of proteins that are involved in cell cycle regulation and/or DNA replication or repair, are primarily thought to be involved in protein-protein interactions. The large (p140) subunit of replication factor C contains a sequence of approximately 100 amino acids in the N-terminal region that binds DNA and is distantly related to known BRCT domains. Here we show that residues 375-480, which include 28 amino acids N-terminal to the BRCT domain, are required for 5'-phosphorylated double-stranded DNA binding. NMR chemical shift analysis indicated that the N-terminal extension includes an alpha-helix and confirmed the presence of a conserved BRCT domain. Sequence alignment of the BRCT region in the p140 subunit of replication factor C from various eukaryotes has identified very few absolutely conserved amino acid residues within the core BRCT domain, whereas none were found in sequences immediately N-terminal to the BRCT domain. However, mapping of the limited number of conserved, surface-exposed residues that were found onto a homology model of the BRCT domain, revealed a clustering on one side of the molecular surface. The cluster, as well as a number of amino acids in the N-terminal alpha-helix, were mutagenized to determine the importance for DNA binding. To ensure minimal structural changes because of the introduced mutations, proteins were checked using one-dimensional (1)H NMR and CD spectroscopy. Mutation of weakly conserved residues on one face of the N-terminal alpha-helix and of residues within the cluster disrupted DNA binding, suggesting a likely binding interface on the protein.     

Genetic analysis of type-specific antigenic determinants of herpes simplex virus glycoprotein C.

Herpes simplex virus type 1 (HSV-1) glycoprotein C (gC-1) elicits a largely serotype-specific immune response directed against previously described determinants designated antigenic sites I and II. To more precisely define these two immunodominant antigenic regions of gC-1 and to determine whether the homologous HSV-2 glycoprotein (gC-2) has similarly situated antigenic determinants, viral recombinants containing gC chimeric genes which join site I and site II of the two serotypes were constructed. The antigenic structure of the hybrid proteins encoded by these chimeric genes was studied by using gC-1- and gC-2-specific monoclonal antibodies (MAbs) in radioimmunoprecipitation, neutralization, and flow cytometry assays. The results of these analyses showed that the reactivity patterns of the MAbs were consistent among the three assays, and on this basis, they could be categorized as recognizing type-specific epitopes within the C-terminal or N-terminal half of gC-1 or gC-2. All MAbs were able to bind to only one or the other of the two hybrid proteins, demonstrating that gC-2, like gC-1, contains at least two antigenic sites located in the two halves of the molecule and that the structures of the antigenic sites in both molecules are independent and rely on limited type-specific regions of the molecule to maintain epitope structure. To fine map amino acid residues which are recognized by site I type-specific MAbs, point mutations were introduced into site I of the gC-1 or gC-2 gene, which resulted in recombinant mutant glycoproteins containing one or several residues from the heterotypic serotype in an otherwise homotypic site I background. The recognition patterns of the MAbs for these mutant molecules demonstrated that (i) single amino acids are responsible for the type-specific nature of individual epitopes and (ii) epitopes are localized to regions of the molecule which contain both shared and unshared amino acids. Taken together, the data described herein established the existence of at least two distinct and structurally independent antigenic sites in gC-1 and gC-2 and identified subtle amino acid sequence differences which contribute to type specificity in antigenic site I of gC.