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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
7R01GM088204-05
Application #
8894893
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
2014-08-01
Budget End
2015-07-31
Support Year
5
Fiscal Year
2014
Total Cost
$285,376
Indirect Cost
$82,376
Name
Massachusetts Institute of Technology
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
001425594
City
Cambridge
State
MA
Country
United States
Zip Code
02142
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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