NMR-Based Conformational Ensembles Explain pH-Gated Opening and Closing of OmpG Channel

Abstract

The outer membrane protein G (OmpG) is a monomeric 33 kDa 14-stranded β-barrel membrane protein functioning as a non-specific porin for the uptake of oligosaccharides in E. coli. Two different crystal structures of OmpG obtained at different values of pH suggest a pH-gated pore opening mechanism. In these structures, extracellular loop 6 extends away from the barrel wall at neutral pH, but is folded back into the pore lumen at low pH, blocking transport through the pore. Loop 6 was invisible in a previously published solution NMR structure of OmpG in DPC micelles, presumably due to conformational exchange on an intermediate NMR time-scale. Here we present an NMR paramagnetic relaxation enhancement (PRE)-based approach to visualize the conformational dynamics of loop 6 and to calculate conformational ensembles that explain the pH-gated opening and closing of the OmpG channel. The different loop conformers detected by the PRE ensemble calculations were validated by disulfide cross-linking of strategically engineered cysteines and electrophysiological single channel recordings. The results indicate a more dynamically regulated channel opening and closing than previously thought and reveal additional membrane-associated conformational ensembles at pH 6.3 and 7.0. We anticipate this approach to be generally applicable to detect and characterize functionally important conformational ensembles of membrane proteins.

Mechanism of OmpG pH-Dependent Gating from Loop Ensemble and Single Channel Studies

“…These findings are consistent with the overall pattern of decreasing open probability with decreasing pH that was empirically observed in this study and elsewhere. 7,15 …”

“…Nuclear magnetic resonance studies of OmpG have shown a much shorter barrel and longer, more disordered extracellular loops that could also invade into the lumen. 13,15 To test if the conformation of loop 6 is the dominant contributor to the gating, a quiet OmpG mutant (qOmpG) was created in which loop 6 was tethered to a neighboring strand to prevent it from bending into the lumen. 14 At pH 8.5, the gating activity of qOmpG was reduced by 95% compared to the wild-type protein.…”

“…Although the mechanism of repulsion between two histidines appears plausible for pH-dependent gating, it cannot explain the fact that gating signals are also observed at neutral and high pH, where repulsion would not occur. In addition, NMR studies indicate that other loops may affect the dynamics of loop 6, 15 although it is unclear how loop-loop interactions may modulate the movement of loop 6 in a pH-dependent manner.…”

Membrane topology of NS2B of dengue virus revealed by NMR spectroscopy

“…5). Signal overlap is always observed in the 1 H-15 N-HSQC spectrum for α-helical membrane proteins [52]. Residue-specific 15 N-labeling of a protein has been proven to be useful in structural and dynamic analysis of proteins [53].…”

Biophysical and biochemical studies of the outer membrane proteins OprG and OprH from Pseudomonas aeruginosa

“…58 Alternatively or in addition, the observed dynamics of extracellular loops 2 and 3 may contribute to amino acid translocation by providing chaperoning environments in the membrane. Concerted loop motions have been shown to contribute to the function of another β-barrel OM protein [133].Unfortunately the P66A, P91A, and double Pro mutants did not stably insert into lipid bilayers to assess the effects of these potentially interesting mutations on the amino acid translocation activity of OprG. These proteins also gave NMR spectra of much poorer quality, which prevented us from determining changes in their dynamic structures, which might have offered additional clues on the mechanism of amino acid transport by OprG.…”