Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis

Abstract

PorB proteins constitute the vast majority of channels in neisserial outer membranes and can be subdivided within meningococcal strains into two distinct and mutually exclusive families that are designated as class 2 and class 3 proteins. We recently characterized the functional activity and conformational stability of a PorB class 2 protein from Neisseria meningitidis (Minetti, C. A. S. A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S. M., and Remeta, D. P. (1997) J. Biol. Chem. 272, 10710 -10720). To evaluate the structure-function relatedness among the PorB proteins, we have employed a combination of electrophoretic and spectroscopic techniques to assess the conformational stability of zwittergent-solubilized class 3 trimers. The functional, physicochemical, and structural properties of the meningococcal class 2 and class 3 proteins are comparable with the notable exception that the latter exhibits a significantly higher susceptibility to SDS. The SDS-induced dissociation and partial unfolding of PorB class 3 is characterized by a single two-state transition with a midpoint at 0.35% SDS. The native trimeric assembly dissociates reversibly, forming partially folded monomers that retain the characteristic ␤-sheet content of the transmembrane domain with a concomitant increase in random coil structure arising from unfolding the rigid surface loops. These results provide new insight into the elucidation of porin folding pathways and the factors that govern the overall structural stability of meningococcal proteins.Bacterial porins are integral outer membrane channel-forming proteins that function as molecular sieves by mediating the exchange of nutrients and waste products with the environment (1, 2). The majority of these porins assemble as trimers of a single polypeptide and exhibit an unusually high resistance to harsh conditions such as low pH, elevated temperatures, and the presence of detergents. As a consequence of such properties, porin oligomeric structures may be visualized in SDS-PAGE.1 Elucidation of the crystal structures of bacterial porins from Rhodobacter capsulatus (3), Rhodopseudomonas blastica (4), and the Escherichia coli porins OmpF, PhoE (5), and LamB (6) reveals the characteristic trimeric structure, each subunit consisting of 16 -18 antiparallel ␤-strands. Recent studies employing circular dichroism spectroscopy indicate that the meningococcal PorB class 2 protein retains physicochemical properties similar to other porins, including the predominance of ␤-sheet structure and retention of native trimeric conformation at high temperature or increased concentrations of denaturants (7).Meningococcal class 2 and class 3 proteins are gene products of two alleles of the single-copy porB gene locus (8) and are thus mutually exclusive within different Neisseria meningitidis strains (9, 10). Among the various class 2 and class 3 proteins sequenced to date, similarities in primary structure are on the order of 60 -70%. The complete amino acid sequences of N. meningitidis PorB class 3 proteins der...

Structural characterization of the fusobacterial non-specific porin FomA suggests a 14-stranded topology, unlike the classical porins

“…The CD spectra strongly suggest that the FomA protein is rich in β-sheet structure and are similar to those obtained with other porins from E. coli (Rosenbusch, 1974) and from Neisseria meningitidis (Minetti et al, 1997(Minetti et al, , 1998. Furthermore, the thermal melting profiles suggest that the FomA protein possesses a β-barrel architecture typical for OMPs (Schulz, 2000), since the cooperative unfolding transition occurs at high temperatures ( 80 mC), reflecting the very stable three-dimensional structures of these proteins.…”

“…Several porins are completely resistant to tryptic hydrolysis also when solubilized (Minetti et al, 1998 ;Rosenbusch, 1974). Since the 37 kDa C-terminal fragment of the FomA protein was completely trypsin resistant when situated in the OM (Bakken et al, 1989a ;Jensen et al, 1996), trypsin digestion experiments were applied to the purified FomA proteins to assess the tertiary structure.…”

Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae : Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations

“…The approximate exclusion limit of each channel was defined as the molecular weight of a sugar that permeated at 10% of the rate of arabinose permeation. The apparent exclusion limits were between 360 and 400 Da for each of the PIBs, which were similar to the ϳ400-Da solute exclusion limits observed for N. meningitidis PorB proteins (21,22). Therefore, mutations in PIB proteins do not appear to decrease pore size.…”

“…Previous studies of porins from N. gonorrhoeae, N. meningitidis, and other bacterial species have demonstrated that recombinant porins expressed in inclusion bodies and refolded in vitro are structurally and functionally indistinguishable from native porins (21,22,31,36). Thus, we purified and refolded gonococcal PIB proteins in a similar manner.…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…The porB locus is occupied in any given strain by either of the two alleles, each expressing either class 2 or class 3 PorB protein, which are only 60 to 70% identical. The class 3 porin, which has extensive alterations in the external-loop sequence (including many short deletions), exhibits the interesting property that its trimers are unusually labile in the presence of SDS even at room temperature (418), producing monomers that apparently still retain the ␤-barrel structure but are altered (denatured) in the loop structure. In this connection, we note that with the OmpF porin of E. coli, modification of loop 2, which connects the neighboring monomers, can make even this porin susceptible to SDS-induced dissociation at lower temperatures (493).…”