# Structures and Dynamics of Proton and Cation-Dependent Channels and Transporters

> **NIH NIH R01** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2022 · $304,748

## Abstract

Project Summary
 This proposal aims to elucidate the structure and mechanism of action of three ion channels and
transporters of viruses and bacteria. Pathogenic organisms use their membrane-bound ion channels
and transporters for survival. Molecular structural information about these membrane proteins forms the
basis for rational design of antiviral and antibiotic compounds to fight and prevent viral and bacterial
infections. We propose to 1) determine the structure of the SARS-CoV-2 envelope (E) protein, which
assembles into a cation-selective channel that stimulates the host inflammasome; 2) investigate the
structural mechanism of the influenza M2 protein, which forms an acid-activated tetrameric proton
channel for influenza virus uncoating; 3) determine the structure of a multidrug-resistant bacterial
transporter, EmrE, to elucidate the mechanisms of proton-coupled substrate transport. These
membrane proteins – E, M2, and EmrE – are drug targets to curb the COVID-19 pandemic, influenza
infections, and antibiotic resistance. In Aim 1 we will investigate the structural basis of the proton
conduction direction in M2 proteins by examining an influenza B M2 (BM2) mutant. Wild-type (WT) AM2
conducts protons only inward, like a transporter, while WT BM2 conducts protons bidirectionally, like a
canonical channel. This difference is correlated with recent data that AM2 undergoes alternating-access
motions to activate while BM2 undergoes a scissor-like motion to activate. To understand these
differences, we will study a BM2 mutant that recapitulates the AM2 inward-rectifying phenotype. We will
measure its structure and dynamics using multidimensional solid-state NMR spectroscopy and correlate
the structural information with channel activities. In Aim 2 we will determine the SARS-CoV-2 E protein’s
transmembrane (TM) structure in lipid bilayers. We will investigate the E structures under different cation
concentrations, pH and with a bound inhibitor, to understand how E conducts cations and how the
conductance can be blocked. 2D and 3D correlation solid-state NMR experiments will be carried out in
conjunction with channel activity measurement. In Aim 3 we will investigate the conformation and
membrane interaction of the cytoplasmic region of E by 31P and 13C NMR, to address the mechanism of
action of the second function of the E protein, which is mediating virus budding and release. In Aim 4,
we will investigate EmrE, which effluxes cationic drugs in a proton-coupled manner to cause antibiotic
resistance in E. coli. We will employ multidimensional 19F NMR techniques to measure protein-drug
distances to constrain the structure of the substrate-binding pocket. These studies should provide
detailed structural insights into the mechanism of membrane transport in some of the most devastating
viruses and bacteria, and should establish the basis for drug design to improve human health.

## Key facts

- **NIH application ID:** 10491987
- **Project number:** 5R01GM088204-12
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Mei Hong
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $304,748
- **Award type:** 5
- **Project period:** 2009-09-30 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10491987

## Citation

> US National Institutes of Health, RePORTER application 10491987, Structures and Dynamics of Proton and Cation-Dependent Channels and Transporters (5R01GM088204-12). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10491987. Licensed CC0.

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