The M2 protein of influenza A and B viruses (AM2 and BM2) forms an acid-activated proton (H+) channel for virus entry and mediates membrane scission in a cholesterol-dependent fashion for virus budding. AM2 is inhibited by the amantadine class of antiviral drugs until the recent emergence of drug-resistant M2 mutants among circulating flu viruses, and no antiviral drugs are yet available against BM2. Thus, structural and mechanistic studies of M2 are important for designing new M2 inhibitors to curb seasonal and pandemic flu. Due to its modular nature and its small size, the M2 protein also serves as a model system for understanding the structural principles governing H+ transport in ion channels and the mechanism of membrane- curvature induction by proteins. So far, the structural basis for how M2 prevents reverse H+ current from the C-terminus to the N-terminus is not yet known. How the N-terminal ectodomain and the C-terminal cytoplasmic tail modulate drug-sensitive H+ conduction through the transmembrane (TM) pore and induce membrane curvature is poorly understood. Structural information about M2 interaction with cholesterol is scarce. Finally, the structure of influenza BM2 in lipid bilayers has not been investigated, and mechanistic information about how BM2 conducts protons is sparse. We propose to employ solid-state NMR spectroscopy to answer these structural and mechanistic questions about influenza AM2 and BM2 in phospholipid bilayers.
In Aim 1, we will investigate the H+ conduction dynamics and drug binding equilibrium of fully functional AM2 containing the ectodomain and the cytoplasmic tail. 2D correlation experiments that detect both TM and extra-membrane residues and 2H NMR experiments that probes drug orientation and dynamics will be performed.
In Aim 2, we will investigate the structure, dynamics and H+ conduction mechanism of BM2. The sidechain conformation and inter-residue contacts of His and Trp in the conserved HxxxW motif will be measured, and hydration of the channel residues will be investigated using 1H-13C correlation experiments.
In Aim 3, we will study gating-deficient mutants of AM2 to understand how Trp41 and Asp44 ensure unidirectional H+ flow from the N-terminus to the C-terminus. Sidechain conformation, dynamics, and inter-residue distances among the key functional residues will be measured.
In Aim 4, we will probe M2-cholesterol interactions by measuring cholesterol orientation and dynamics, cholesterol-induced chemical shift changes, and intermolecular distances to constrain the putative M2-cholesterol complex.

Public Health Relevance

The M2 protein of influenza viruses forms a drug-targetable proton channel and mediates virus budding, thus high-resolution structural studies of M2 have high public health significance. We will use solid-state NMR spectroscopy to elucidate the structural basis of M2's proton channel activity and M2 interaction with cholesterol, to guide rational design of new antiviral drugs to prevent flu pandemics and advance our fundamental understanding of ion channels and virus budding.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM088204-08
Application #
9417014
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Ainsztein, Alexandra M
Project Start
2009-09-30
Project End
2021-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
8
Fiscal Year
2018
Total Cost
Indirect Cost
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
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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
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
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
Kwon, Byungsu; Hong, Mei (2016) The Influenza M2 Ectodomain Regulates the Conformational Equilibria of the Transmembrane Proton Channel: Insights from Solid-State Nuclear Magnetic Resonance. Biochemistry 55:5387-97
Williams, Jonathan K; Tietze, Daniel; Lee, Myungwoon et al. (2016) Solid-State NMR Investigation of the Conformation, Proton Conduction, and Hydration of the Influenza B Virus M2 Transmembrane Proton Channel. J Am Chem Soc 138:8143-55
Elkins, Matthew R; Wang, Tuo; Nick, Mimi et al. (2016) Structural Polymorphism of Alzheimer's ?-Amyloid Fibrils as Controlled by an E22 Switch: A Solid-State NMR Study. J Am Chem Soc 138:9840-52
Liang, Ruibin; Swanson, Jessica M J; Madsen, Jesper J et al. (2016) Acid activation mechanism of the influenza A M2 proton channel. Proc Natl Acad Sci U S A :
Liao, Shu Y; Lee, Myungwoon; Wang, Tuo et al. (2016) Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location. J Biomol NMR 64:223-37
Williams, Jonathan K; Schmidt-Rohr, Klaus; Hong, Mei (2015) Aromatic spectral editing techniques for magic-angle-spinning solid-state NMR spectroscopy of uniformly (13)C-labeled proteins. Solid State Nucl Magn Reson 72:118-26

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