The influenza M2 protein forms a pH-activated proton channel that is essential for the virus lifecycle. Inhibition of the H+ channel activity by the amantadine class of antiviral drugs has been made ineffective by mutations in the M2 transmembrane domain. High-resolution structure determination of M2 is thus paramount for developing new antiviral drugs to target amantadine- resistant M2 variants. This small protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and is thus an excellent model system for understanding larger voltage-gated H+ channels and pH-gated ion channels. Work funded by this research proposal has previously led to the elucidation of the pharmacologically relevant drug-binding site in M2 and revealed pH-dependent dynamics of the proton-selective residue, His37. However, new H+ conduction models have since been proposed, and the structure basis for channel gating by Trp41 has not been studied.
Aim 1 of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at acidic pH when the channel is activated. H-bonding, protonation/deprotonation dynamics, and the effects of inhibitors on His37 structure will be measured. Both amantadine and Cu2+ will be used as inhibitors.
Aim 2 is to elucidate Trp41 structure and interaction with His37 by measuring chemical shifts and inter-atomic distances. In addition to the H+ channel activity, M2 mediates virus budding by causing membrane curvature in a cholesterol-dependent fashion. We will investigate M2-membrane and M2-cholesterol interactions by distance and relaxation NMR measurements.
Aim 3 will measure whether M2 preferentially binds to highly curved regions of the membrane. M2 interacts with the matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is yet available for the cytoplasmic domain.
In aim 4 we will determine the 3D structure of full-length M2 in lipid bilayers using multidimensional MAS correlation experiments.

Public Health Relevance

The influenza virus M2 protein is the target of antiviral drugs but has evolved to evade it. This project seeks to elucidate the atomic structure of the M2 protein in the hope that this knowledge will lead to new antiviral drugs to combat future flu pandemics. Elucidating the molecular basis for the proton-channel activity of the protein may also give insights into the other proton channels in immune-system white blood cells.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BCMB-R (02))
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Chin, Jean
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Massachusetts Institute of Technology
Schools of Arts and Sciences
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Gelenter, Martin D; Hong, Mei (2018) Efficient 15N-13C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination. J Phys Chem B 122:8367-8379
Elkins, Matthew R; Sergeyev, Ivan V; Hong, Mei (2018) Determining Cholesterol Binding to Membrane Proteins by Cholesterol 13C Labeling in Yeast and Dynamic Nuclear Polarization NMR. J Am Chem Soc 140:15437-15449
Shcherbakov, Alexander A; Hong, Mei (2018) Rapid measurement of long-range distances in proteins by multidimensional 13C-19F REDOR NMR under fast magic-angle spinning. J Biomol NMR 71:31-43
Mandala, Venkata S; Gelenter, Martin D; Hong, Mei (2018) Transport-Relevant Protein Conformational Dynamics and Water Dynamics on Multiple Time Scales in an Archetypal Proton Channel: Insights from Solid-State NMR. J Am Chem Soc 140:1514-1524
Roos, Matthias; Mandala, Venkata S; Hong, Mei (2018) Determination of Long-Range Distances by Fast Magic-Angle-Spinning Radiofrequency-Driven 19F-19F Dipolar Recoupling NMR. J Phys Chem B 122:9302-9313
Mandala, Venkata S; Williams, Jonathan K; Hong, Mei (2018) Structure and Dynamics of Membrane Proteins from Solid-State NMR. Annu Rev Biophys 47:201-222
Liao, Shu Y; Lee, Myungwoon; Hong, Mei (2018) Interplay between membrane curvature and protein conformational equilibrium investigated by solid-state NMR. J Struct Biol :
Mandala, Venkata S; Liao, Shu-Yu; Kwon, Byungsu et al. (2017) Structural Basis for Asymmetric Conductance of the Influenza M2 Proton Channel Investigated by Solid-State NMR Spectroscopy. J Mol Biol 429:2192-2210
Dai, Peng; Williams, Jonathan K; Zhang, Chi et al. (2017) A structural and mechanistic study of ?-clamp-mediated cysteine perfluoroarylation. Sci Rep 7:7954
Williams, Jonathan K; Shcherbakov, Alexander A; Wang, Jun et al. (2017) Protonation equilibria and pore-opening structure of the dual-histidine influenza B virus M2 transmembrane proton channel from solid-state NMR. J Biol Chem 292:17876-17884

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