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

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 wateraccessible 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. [ABSTRACT FROM AUTHOR]