Structure, Dynamics, and Substrate Specificity of the OprO Porin from Pseudomonas aeruginosa

Abstract

The outer membrane (OM) of Gram-negative bacteria functions as a selective permeability barrier between cell and environment. For nutrient acquisition, the OM contains a number of channels that mediate uptake of small molecules by diffusion. Many of these channels are specific, i.e., they prefer certain substrates over others. In electrophysiological experiments, the OM channels OprP and OprO from Pseudomonas aeruginosa show a specificity for phosphate and diphosphate, respectively. In this study we use x-ray crystallography, free-energy molecular dynamics (MD) simulations, and electrophysiology to uncover the atomic basis for the different substrate specificity of these highly similar channels. A structural analysis of OprP and OprO revealed two crucial differences in the central constriction region. In OprP there are two tyrosine residues, Y62 and Y114, whereas the corresponding residues in OprO are phenylalanine F62 and aspartate D114. To probe the importance of these two residues in generating the different substrate specificities, the double mutants were generated in silico and in vitro. Applied-field MD simulations and electrophysiological experiments demonstrated that the double mutations interchange the phosphate and diphosphate specificities of OprP and OprO. Our findings outline a possible strategy to rationally design channel specificity by modification of a small number of residues that may be applicable to other pores as well.

Fosfomycin Permeation through the Outer Membrane Porin OmpF

“…The obtained results will be most interesting for studies of other porins with similar positively charged arginine ladders. Among these porins are the OmpF homologous or semihomologous OM Gram-negative bacterial proteins OmpC (68), PhoE (26), Omp32 (69), OmpK35/36 (70), OmpE35/ 36 (71), OprD (72), OprP (73), and OprO (74). Similar mechanisms as extracted here will likely apply, but especially for the narrower pores like OprD and OprP, the permeation rates will be much smaller than those for OmpF.…”

Exposure of Bacterial Biofilms to Electrical Current Leads to Cell Death Mediated in Part by Reactive Oxygen Species

“…Although up-regulation may be due to the limited phosphate in the buffer, comparisons were done against control biofilms receiving no DC, leading us to believe that additional stress is being applied to the treated biofilms. Additionally, an increase in the phosphate-specific porin OprO transcript suggests that bacteria are actively trying to transport phosphate during exposure to DC [27]. It has also been proposed that under P i limiting conditions, catalase is induced by P .…”

Iron oxidation by a fused cytochrome-porin common to diverse iron-oxidizing bacteria

“…4 ) and homology matching by HHpred (Söding et al , 2005). This C-terminal section has low sequence homology, but high structural homology to the outer membrane phosphate-selective porins OprO and OprP (PDB structures 4RJW and 2O4V (Moraes et al , 2006; Modi et al , 2015); Supplemental Table 1 ). Poor sequence conservation is typical of porins (Nikaido, 2003), and since the porin portion constitutes most of the sequence, this explains why Cyc2 homologs tend to have low amino acid identity ( Supplemental Fig.…”