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
Williams, Jonathan K; Hong, Mei (2014) Probing membrane protein structure using water polarization transfer solid-state NMR. J Magn Reson 247:118-27
Lee, Myungwoon; Hong, Mei (2014) Cryoprotection of lipid membranes for high-resolution solid-state NMR studies of membrane peptides and proteins at low temperature. J Biomol NMR 59:263-77
Joh, Nathan H; Wang, Tuo; Bhate, Manasi P et al. (2014) De novo design of a transmembrane ZnĀ²?-transporting four-helix bundle. Science 346:1520-4
Williams, Jonathan K; Tietze, Daniel; Wang, Jun et al. (2013) Drug-induced conformational and dynamical changes of the S31N mutant of the influenza M2 proton channel investigated by solid-state NMR. J Am Chem Soc 135:9885-97
Hong, Mei; Schmidt-Rohr, Klaus (2013) Magic-angle-spinning NMR techniques for measuring long-range distances in biological macromolecules. Acc Chem Res 46:2154-63
Fritzsching, K J; Yang, Y; Schmidt-Rohr, K et al. (2013) Practical use of chemical shift databases for protein solid-state NMR: 2D chemical shift maps and amino-acid assignment with secondary-structure information. J Biomol NMR 56:155-67
Liao, Shu Yu; Fritzsching, Keith J; Hong, Mei (2013) Conformational analysis of the full-length M2 protein of the influenza A virus using solid-state NMR. Protein Sci 22:1623-38
Williams, Jonathan K; Zhang, Yuan; Schmidt-Rohr, Klaus et al. (2013) pH-dependent conformation, dynamics, and aromatic interaction of the gating tryptophan residue of the influenza M2 proton channel from solid-state NMR. Biophys J 104:1698-708
Su, Yongchao; Hu, Fanghao; Hong, Mei (2012) Paramagnetic Cu(II) for probing membrane protein structure and function: inhibition mechanism of the influenza M2 proton channel. J Am Chem Soc 134:8693-702
Wang, Tuo; Cady, Sarah D; Hong, Mei (2012) NMR determination of protein partitioning into membrane domains with different curvatures and application to the influenza M2 peptide. Biophys J 102:787-94

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