# Solid-state NMR of the influenza M2 protein in lipid bilayers

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2020 · $358,544

## Abstract

Project Summary
 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.

## Key facts

- **NIH application ID:** 9857618
- **Project number:** 5R01GM088204-10
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Mei Hong
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $358,544
- **Award type:** 5
- **Project period:** 2009-09-30 → 2021-09-19

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9857618

## Citation

> US National Institutes of Health, RePORTER application 9857618, Solid-state NMR of the influenza M2 protein in lipid bilayers (5R01GM088204-10). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9857618. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*