Amilorides Bind to the N-Terminal Transmembrane Helix of the Sars-CoV-2 Envelope Protein

SARS-CoV-2, the cause of the COVID-19 pandemic is one of the most dangerous threats to humanity in recent history. At the onset of this global crisis, scientists quickly turned to vaccine development as the primary focus of research, while drug discovery and development has trailed, largely due to the lack of structural information characterizing the free and drug-bound conformations of the viral proteins. To address this, the envelope (E) protein was surveyed as a target for treatment via solution NMR due to its involvement in the virus life cycle and putative ion channel activity. Before beginning to characterize the structure and dynamics of the E protein, a novel expression and purification scheme needed to be developed and optimized. Through construct design and troubleshooting several experimental methods, an effective sample preparation approach was established, which facilitated the implementation of structural studies of the unmodified, full-length E protein and its truncated counterparts. Through CD and solution NMR spectroscopy, the N- and C-terminal domains of the E protein constructs were found to be conserved, which has implications on their abilities to bind drugs. The E protein drug binding site was determined to be within the N-terminal transmembrane domain and the binding affinities of several amiloride compounds with E protein were characterized. The N15A mutation was found to induce significant CSPs for signals from binding site-associated residues, which reflected structural changes resulting in the inability to bind HMA, suggesting that Asn15 plays an essential role in preserving the conformation of E protein’s binding site.