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 mutants. The small M2 protein contains all the machinery necessary for pH activation, H+ selectivity, and gating, and thus also provides an excellent model system for understanding larger and more complex voltage-gated H+ channels and other pH-gated ion channels. Work funded by this research proposal has already 1) led to the elucidation of the pharmacologically relevant drug binding site in M2 and the drug-complexed high-resolution structure in the lipid bilayer, and 2) revealed novel pH-dependent dynamics of the proton-selective residue, His37. However, new alternative H+ conduction models have been proposed in the meantime, and the structure basis for channel gating by Trp41 has not been studied.
The first aim of this proposal is to elucidate the H+ conduction mechanism of M2 by examining His37 structure at mildly acidic pH when the channel is first activated. Sidechain 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, and Cu2+ paramagnetic relaxation enhancement effects will be explored for structure determination.
The second aim i s to elucidate Trp41 structure and interaction with His37 as a function of pH, to understand how these two residues act in unison to achieve channel gating, again in a bilayer environment. In addition to the H+ channel activity, M2 also 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. The hypothesis that M2 preferentially localizes to highly curved regions of the membrane will be tested. Finally, M2 interacts with matrix protein M1 through its cytoplasmic tail during virus assembly and budding. No structural information is available so far for the cytoplasmic domain. We will determine the three-dimensional structure of full-length M2 in lipid bilayers using multidimensional magic-angle-spinning solid-state NMR techniques, to lay the ground for future investigations of the M2-M1 interactions important for the influenza life cycle.

Public Health Relevance

The influenza virus M2 protein is the target of an antiviral drug but has recently 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 M2 protein may also give insights into the inner workings of proton channels of white blood cells that kill bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM088204-03
Application #
8211383
Study Section
Special Emphasis Panel (ZRG1-BCMB-R (02))
Program Officer
Chin, Jean
Project Start
2009-09-30
Project End
2016-07-31
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
3
Fiscal Year
2012
Total Cost
$273,492
Indirect Cost
$70,492
Name
Iowa State University
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
005309844
City
Ames
State
IA
Country
United States
Zip Code
50011
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 :
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
Elkins, Matthew R; Williams, Jonathan K; Gelenter, Martin D et al. (2017) Cholesterol-binding site of the influenza M2 protein in lipid bilayers from solid-state NMR. Proc Natl Acad Sci U S A 114:12946-12951

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