The spontaneous gating activity of OmpC porin is affected by mutations of a putative hydrogen bond network or of a salt bridge between the L3 loop and the barrel

Abstract

Porins are trimeric channel-forming proteins of the outer membrane of Escherichia coli. Each subunit contains 16 beta-strands forming a transmembrane beta-barrel whose pore is constricted by the third extracellular loop (L3). We investigated the effects of site-directed mutations at two critical regions of the OmpC porin: (i) the D315A mutation targets a key component of a putative hydrogen bond network linking the L3 loop to the adjacent barrel wall and (ii) the D118Q, R174Q and R92Q mutations target putative salt bridges at the root of the L3 loop. We purified the outer membrane fractions obtained from each mutant and reconstituted them in liposomes suitable for electrophysiology. Patch clamp experiments showed that the frequency of spontaneous transitions between open and closed states is increased in the D315A, D118Q and R92Q mutants but unchanged in the R174Q mutant. These transitions are not driven by transmembrane voltage changes and represent the thermal oscillations between functionally distinct conformations. The asymmetric voltage-dependent inactivation of the channels is not affected by the mutations, however, suggesting different molecular mechanisms for the spontaneous and voltage-dependent gating processes. We propose that the positioning or flexibility of the L3 loop across the pore, as governed by the putative hydrogen-bond network and a salt bridge, play a role in determining the frequency of spontaneous channel gating.

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

“…The electrophysiological properties of these mutant porins were examined by patch clamp of liposome blisters containing multiple porin channels. Some of the mutant porins displayed a dramatic increase in the closing rate and decrease in open probability relative to wildtype OmpC (17,18). These changes were interpreted to be the result of changes in the flexibility of loop 3 within the barrel, caused by the loss of hydrogen bonds and/or salt bridges between loop 3 and the outer barrel.…”

“…The increased channel noise and increased time spent in subconductance states observed in the variant PIBs are reminiscent of the changes in the channel properties of the OmpC porin with mutations designed to disrupt the interaction of loop 3 with the outer barrel (17,18). The electrophysiological properties of these mutant porins were examined by patch clamp of liposome blisters containing multiple porin channels.…”

Molecular basis of usher pore gating in Escherichia coli pilus biogenesis

“…Disruption of this salt bridge interaction promotes an open usher state, whereas flipping the residue identities across the two positions maintains electrostatic contact and the WT antibiotic sensitivity phenotype, confirming the critical nature of this salt bridge as part of a mechanism of pore gating. Salt bridges were also shown to be important factors in the gating of much smaller pores without a PLUG domain such as OmpC and OmpA (33,34). Despite increased pore permeability of the R305A mutant, functional pilus biogenesis was not affected.…”

Outer membrane protein G: Engineering a quiet pore for biosensing

“…The gating of OmpA is associated with breaking and rearrangement of the salt bridges in the center of the barrel (21,23). In the case of the trimeric porin OmpC, it has been proposed that spontaneous gating is related to the positioning or flexibility of loop 3, which folds to form an eyelet (constriction zone) within the pore (24,25). It has been previously suggested that, under acidic conditions, L6 is implicated in the pH-dependent gating of OmpG (7).…”