Structures and Dynamics of Proton- and Cation-Conducting Viroporins - Project Summary: Structures and Dynamics of Proton- and Cation-Conducting Viroporins We seek to elucidate the structure, assembly, and mechanism of ion channels in two classes of pandemic-causing viruses, influenza and coronaviruses. These viruses encode viroporins, small membrane proteins that form pores to cause pathogenicity to cells. Elucidating the structures and mechanism of these viroporins is essential for expanding the arsenal of antiviral drugs to treat deadly viral infections and for advancing basic knowledge about the principle of ion conduction by membrane proteins. We will use solid-state NMR (ssNMR) spectroscopy to investigate three viroporins: the influenza B virus M2 (BM2) protein, the SARS-CoV-2 envelope (E) protein, and the human coronavirus OC43 E protein. In Aim 1, we will investigate the structures and mechanisms of polar and aromatic residues that are key to the functions of BM2 and SARS-CoV-2 E. Using ssNMR, we will measure the sidechain conformation and dynamics of the proton-selective histidine and the gating tryptophan in wild-type and mutant BM2 channels. These experiments will reveal how these residues interact to regulate the proton conduction direction in this archetypal proton channel. We will also investigate the ion conduction mechanism of SARS-CoV-2 E by studying two mutants, T9I and N15A. We hypothesize that these mutants disrupt an N-terminal polar network that mediates proton and cation permeation. In Aim 2, we will investigate the oligomeric structure of full-length E of SARS-CoV-2. Biochemical and ssNMR data suggest that E’s oligomerization may be plastic, and this plasticity may allow this multi-functional protein to perform the right function at the right place and time. We will develop a dynamic nuclear polarization sensitivity-enhanced 19F spin diffusion NMR technique to determine oligomer structure distributions of membrane proteins in lipid bilayers and will apply this technique to full-length E. Our recent NMR data showed that E’s transmembrane domain form pentamers that cluster in membranes that contain phosphatidylinositol, an essential lipid for a myriad of cellular functions. In Aim 3, we will investigate the structure of the phosphatidylinositol-E complex by 13C-labeling the lipid using yeast and measuring protein-lipid contacts by ssNMR. These experiments will not only illuminate the structural basis of phosphatidylinositol interaction with E but also pave the way for general studies of protein- lipid interactions. To determine the molecular features of coronavirus E proteins that correlate with pathogenicity, in Aim 4 we will investigate the E protein of OC43, one of the coronaviruses that cause the common cold. Using 2D and 3D ssNMR and nanometer-distance measurement techniques, we will determine the structure of OC43 E’s transmembrane domain. Together, these studies will transform our understanding of the principles of ion conduction by pathogenic virus proteins and establish a basis for designing viroporin inhibitors as antiviral drugs.
