Structure and function of the PorB porin from disseminating Neisseria gonorrhoeae

Abstract

The outer membrane of Gram-negative bacteria contains a large number of channel-forming proteins, porins, for the uptake of small nutrient molecules. Neisseria gonorrhoeae PorBIA (PorB of serotype A) are associated with disseminating diseases and mediate a rapid bacterial invasion into host cells in a phosphate-sensitive manner. To gain insights into this structure-function relationship we analysed PorBIA by X-ray crystallography in the presence of phosphate and ATP. The structure of PorBIA in the complex solved at a resolution of 3.3 Å (1 Å=0.1 nm) displays a surplus of positive charges inside the channel. ATP ligand-binding in the channel is co-ordinated by the positively charged residues of the channel interior. These residues ligate the aromatic, sugar and pyrophosphate moieties of the ligand. Two phosphate ions were observed in the structure, one of which clamped by two arginine residues (Arg92 and Arg124) localized at the extraplasmic channel exit. A short β-bulge in β2-strand together with the long L3 loop narrow the barrel diameter significantly and further support substrate specificity through hydrogen bond interactions. Interestingly the structure also comprised a small peptide as a remnant of a periplasmic protein which physically links porin molecules to the peptidoglycan network. To test the importance of Arg92 on bacterial invasion the residue was mutated. In vivo assays of bacteria carrying a R92S mutation confirmed the importance of this residue for host-cell invasion. Furthermore systematic sequence and structure comparisons of PorBIA from Neisseriaceae indicated Arg92 to be unique in disseminating N. gonorrhoeae thereby possibly distinguishing invasion-promoting porins from other neisserial porins.

Crystallographic analysis of Neisseria meningitidis PorB extracellular loops potentially implicated in TLR2 recognition

“…The variable loop regions (VR) 1 to 4, located in L1, L5, L6 and L7, are used for the classification of N. meningitidis clonal complexes. N. meningitidis PorB (strain W135) and N. gonorrhoeae PorB IA , (strain N242) have been recently crystallized (Tanabe et al, 2010;Zeth et al, 2013), confirming the predicted PorB topology model.…”

“…The model rebuilding was facilitated by the structure of PorB 1A from N. gonorrhoeae strain AN242 (PorB Ng ) (PDB ID: 4AUI) (Zeth et al, 2013). This porin has a 77% sequence identity to PorB WT and rms deviation values for the Cα atoms of 0.99 Å for (271 residues), allowing a significant improvement of loop tracing.…”

Horizontal Bilayer for Electrical and Optical Recordings

“…Figure 12a shows the fluorescence intensity distribution (FIDA) traces with the corresponding fit which calculates the concentration and the brightness of PorB-637 in the buffer (Figure 11b) and the bilayer (Figure 11c), respectively. The changes in the molecular brightness are likely to be attributed to changes of the dielectrical environment of the fluorophore upon integration of PorB into the membrane for the following reasons: PorB forms a stable trimer in detergent-buffer solutions [52,53] and does not change its oligomeric state upon functional incorporation into the membrane [52]. Therefore oligomerization of the protein cannot explain the increase in the molecular brightness of the fluorophore upon integration of the labeled protein.…”

Neisseriae internalization by epithelial cells is enhanced by TLR2 stimulation

“…Porins also favor bacterial survival following host infection by inhibiting phagocyte functions [33, 34] and modulating host cell death [35, 36]. It has been established that criticalr esidues in the surface-exposed loop regions of PorB influence organisms’ internalization by host cells [9, 13, 17] and may be involved in the hyper-invasive features of some NM strains [18]. Our group has recently reported a prevalence of negative surface charges is reported in loops 1, 4, 6 and 7 of PorB from invasive meningococcal serogroup B clinical isolates, while overall positive charges are reported in the corresponding PorB loops of invasive meningococcal serogroup C clinical isolates [19].…”