Transverse relaxation dispersion of the p7 membrane channel from hepatitis C virus reveals conformational breathing

Abstract

The p7 membrane protein encoded by Hepatitis C virus (HCV) assembles into a homo-hexamer that selectively conducts cations. An earlier solution NMR structure of the hexameric complex revealed a funnel-like architecture and suggests that a ring of conserved asparagines near the narrow end of the funnel are important for cation interaction. NMR based drug-binding experiments also suggest that rimantadine can allosterically inhibit ion conduction via a molecular wedge mechanism. These results suggest the presence of dilation and contraction of the funnel tip that are important for channel activity and that the action of the drug is attenuating this motion. Here, we determined the conformational dynamics and solvent accessibility of the p7 channel. The proton exchange measurements show that the cavity-lining residues are largely water accessible, consistent with the overall funnel shape of the channel. Our relaxation dispersion data show that residues Val7 and Leu8 near the asparagine ring are subject to large chemical exchange, suggesting significant intrinsic channel breathing at the tip of the funnel. Moreover, the hinge regions connecting the narrow and wide regions of the funnel show strong relaxation dispersion and these regions are the binding sites for rimantadine. Presence of rimantadine deceases the conformational dynamics near the asparagine ring and the hinge area. Our data provide direct observation of µs – ms dynamics of the p7 channel and support the molecular wedge mechanism of rimantadine inhibition of the HCV p7 channel.

Hepatitis C virus sequence divergence preserves p7 viroporin structural and dynamic features

“…The pattern of amide proton exchange of p7(5a/EUH1480) in DPC was similar to that of p7(1b/J4) in Cyclofos-6, and the results are consistent with the exchange rates previously reported for p7(5a/EUH1480) in similar conditions 30 . Some regions, including the N- and C-termini, helix 1, and H17, exhibited higher exchange rates in p7(5a/EUH1480), which may be due to differences in hydrophobicity between the constructs: The regions including residues 13–20 and 53–58 are more hydrophobic in p7(1b/J4) than p7(5a/EUH1480) (Supplementary Fig.…”

Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors

“…A recent NMR structure suggests a different arrangement of short helical segments of p7, predicting the formation of a binding pocket for rimantadine outside of the channel pore, where it acts as an allosteric inhibitor (23). An NMR-based determination of solvent accessibility confirmed this architecture of the p7 protein and the allosteric mechanism of rimantadine inhibition (28). The activity of p7 as an intracellular pH shunt is believed to be involved in critical steps of the viral replication cycle, controlling acidification of endosomes and virion-loaded particles, and promoting vesicle trafficking and assembly and release of virions (29).…”

A functional NMR for membrane proteins: dynamics, ligand binding, and allosteric modulation

“…Dev et al . performed relaxation dispersion measurement on p7 channel in DPC micelles using 2D CPMG TROSY–HSQC experiment at 600 and 700 MHz . They found that while most of the channel did not show relaxation dispersion, residues at the H1–H2 hinge (Phe19) and the narrow end of the cavity (Val7, Leu8) experienced chemical exchanges ( K ex ∼ 1000 ± 79 s −1 and ∼10% excited state).…”