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

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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.