Structural and Functional Characterization of a Recombinant PorB Class 2 Protein from Neisseria meningitidis

Abstract

An outer membrane PorB class 2 protein from Neisseria meningitidis has been overexpressed in Escherichia coli, isolated from inclusion bodies, and refolded in the presence of zwitterionic detergent. The purified recombinant and native (strain M986) counterpart exhibit most of the typical functional and structural properties that are characteristic of bacterial porins. Channel forming activity has been monitored by incorporating class 2 into reconstituted liposomes and measuring the permeation rates of various oligosaccharides through the proteoliposomes to derive a pore diameter of ϳ1.6 nm. Structural studies employing a combination of spectroscopic and electrophoretic techniques reveal that recombinant and native class 2 are identical in terms of overall conformational stability. Both proteins form stable trimers in zwitterionic detergent and retain significant secondary and tertiary structure in the presence of SDS. The thermal unfolding of zwittergen-solubilized class 2 trimers (T m ‫؍‬ 88°C) is reversible and characterized by solvent exposure of aromatic residues with concomitant disruption of tertiary and partial loss of secondary structures. SDS-induced destabilization and irreversible unfolding of the native trimeric assembly occurs at temperatures above 60°C. Our physicochemical studies of PorB class 2 protein furnish significant insight regarding the structural and functional properties of this meningococcal outer membrane protein within the porin superfamily.Porins are integral outer membrane proteins that function as molecular sieves in Gram-negative bacteria, mediating the aqueous diffusion of solutes through the water-filled channels derived from their unique folding/assembly (1-2). Although meningococcal outer membranes may contain as many as five major proteins, much of the observed serotype specificity can be attributed to PorB class 2 and class 3 proteins, both of which are mutually exclusive within different meningococcal strains and alleles from the porB gene (3). Studies on meningococcal outer membranes (3, 4) originally suggested that class 2 protein might function as a bacterial porin on the basis of noted similarities with Escherichia coli protein I (5). The strongest argument to date regarding porin function arises from comparative studies with other neisserial strains (i.e. gonococcal) for which equivalents of class 2 and class 3 proteins have been proposed (6). Evidence regarding the porin function of protein I from Neisseria gonorrhoeae has been furnished by Douglas et al. (7) and extended through further biophysical characterization (8 -9). Meningococcal porin function has also been assessed in vivo by characterizing the relative sensitivity of native and mutant strains to hydrophilic antibiotics (10 -11). A remarkable finding that appears exclusive to pathogenic neisserial strains (including the class 2-expressing serotypes) is the spontaneous transfer of porin molecules from the outer membrane of whole cells into artificial lipid bilayers (12) as well as target cell membranes (13)(1...

The PorB porin from commensal Neisseria lactamica induces Th1 and Th2 immune responses to ovalbumin in mice and is a potential immune adjuvant

“…We have supporting evidence that the presumed native trimeric structure of Nlac PorB is maintained upon purification, suggested by the presence of high molecular weight bands in non-denaturing SDS PAGE, in a similar manner to PorB from N. meningitidis [56]. In fact, a high degree of structural and functional similarities between neisserial porins has been described [51]; they share a high content of β-pleated sheet, a predicted 16-strand β-barrel fold and multiple surface-exposed, variable, hydrophilic loops [51,67]. However, an aminoacid sequence alignment between PorB from N. meningitidis and N. lactamica has determined that the regions of homology do not include some surface exposed loops (loop I, IV, V and VI) [51].…”

Neisseria meningitidisporin PorB interacts with mitochondria and protects cells from apoptosis

“…The structure of neisserial porins reveals a trimeric ␤-pleated barrel with a high percentage (36%) of ␤-sheet secondary structures (39,40). Mitochondrial porins, or VDACs, also are characterized by a similar ␤-barrel conformation and share functional characteristics with neisserial porins, including regulation of the pore size by nucleotides and the ability of ''autodirected'' insertion into phospholipid membranes (34).…”

Structural basis for solute transport, nucleotide regulation, and immunological recognition of Neisseria meningitidis PorB

“…Similar to other Gram-negative bacteria, solute translocation across the outer membrane is essential for neisserial survival. In N. meningitidis, PorB is the second most prevalent outer membrane protein (OMP), and has previously been characterized as a voltage-gated nonselective porin (2) that translocates smaller sugars faster than larger sugars (3).…”