Conformational Changes of an Ion Channel Detected Through Water−Protein Interactions Using Solid-State NMR Spectroscopy

Abstract

The influenza A virus M2 protein is a pH-gated and amantadine-inhibited proton channel important for the virus life cycle. Proton conduction by M2 is known to involve water, however direct experimental evidence of M2-water interaction is scarce. Using 1H spin diffusion solid-state NMR, we have now determined the water accessibility of the M2 transmembrane domain (M2-TM) in virus-envelope-mimetic lipid membranes and its changes with environment. Site-specific water-protein magnetization transfer indicates that, in the absence of amantadine, the initial spin diffusion rate mainly depends on the radial position of the residues from the pore: pore-lining residues along the helix have similarly high water accessibilities compared to lipid-facing residues. Upon drug binding, the spin diffusion rates become much slower for Gly34 in the middle of the helix than for the N-terminal residues, indicating that amantadine is bound to the pore lumen between Gly34 and Val27. Water-protein spin diffusion buildup curves indicate that spin diffusion is the fastest in the low-pH open state, slower in the high-pH closed state, and the slowest in the high-pH amantadine-bound state. Simulations of the buildup curves using a 3D lattice model yielded quantitative values of the water-accessible surface area and its changes by pH and drug binding. These data provide direct experimental evidence of the pH-induced change of the pore size and the drug-induced dehydration of the pore. This study demonstrates the capability of 1H spin diffusion NMR for elucidating water interactions with ion channels, water pores, and proton pumps, and for probing membrane protein conformational changes that involve significant changes of water-accessible surface areas.

Tidal surge in the M2 proton channel, sensed by 2D IR spectroscopy

“…The pH dependence and the ultrafast relaxation arising from fluctuations of the water suggests that the number of water molecules in the channel has significantly increased at pH 6 compared with pH 8. Recent spin diffusion NMR results (37) indicate that residues between Val27 and Gly34 are more accessible to water at low pH than at high pH and that the water is less mobile (on a millisecond timescale) at high pH. These observations are consistent with the 2D IR results.…”

“…The waiting time dependence of the 2D IR spectra indicates that the frequency of the vibrator is undergoing equilibrium fluctuations as exchange occurs between accessible equilibrium conformational states on the measured timescales (3). The SSNMR spectra, many of which are time averaged, also change with pH (25,37,39). The 2D IR spectrum gives information and dynamics on timescales as short as a few hundred femptoseconds, allowing one to probe configurations that would be averaged on the nanosecond to second NMR timescales.…”

“…An additional pair of waters bridge the opposite face of the His37 tetrad, forming hydrogen bonds to the N ε of the imidazoles. MD simulations suggest that this structure is partly maintained at room temperature; also solid-state NMR (SSNMR) (32,37) of the TM domain of M2 in phospholipid bilayers shows that in frozen water the histidine-water hydrogen bonding is highly responsive to the degree of protonation of the four His37 residues. Water above the histidines (in the sense of Fig.…”

High-resolution structures of the M2 channel from influenza A virus reveal dynamic pathways for proton stabilization and transduction

“…A previously solved 1.65-Å crystal structure (9) showed six ordered waters immediately above the His37 tetrad, but ordered waters spanning the entire aqueous pore of M2 have not been observed until now. Previous MD simulations suggested a pore with mobile waters (12,15), whereas the results of NMR and IR experiments are more consistent with an environment that is more similar to bulk water at low pH (13,19,20). However, it is difficult to deconvolute the changes in the water structure and dynamics when the protonation of His37 is raised from those induced indirectly via the conformation of the protein's main chain.…”

“…M2 is activated at low pH by protonation of His37, which also participates in proton conduction by shuttling protons into the interior of the virus (5-7). His37 lies near the center of the bilayer, where it is connected to the viral exterior by a water-filled pore through which protons must pass to gain access to the viral interior (8)(9)(10)(11)(12)(13).…”