Polar Networks Mediate\nIon Conduction of the SARS-CoV‑2\nEnvelope Protein

The SARS-CoV-2 E protein conducts cations across the\ncell membrane\nto cause pathogenicity to infected cells. The high-resolution structures\nof the E transmembrane domain (ETM) in the closed state at neutral\npH and in the open state at acidic pH have been determined. However,\nthe ion conduction mechanism remains elusive. Here, we use solid-state\nNMR spectroscopy to investigate the side chain structure, dynamics,\nand interactions of five polar residues at the N-terminal entrance\nof the channel and three polar residues at the C-terminal end. The\nchemical shifts of the N-terminal Glu8 reveal that the Glu side chain\ninteracts with protons, Ca<sup>2+</sup> and two neighboring Thr residues,\nand adopts distinct motionally averaged conformational ensembles.\nThese polar interactions are sensitive to the presence of negatively\ncharged lipids in the membrane. A T9I mutation, prevalent in the Omicron\nvariants of SARS-CoV-2 E, perturbs these interactions and partially\nimmobilizes the N-terminal segment. Deeper into the channel, two polar\nresidues, Asn15 and Ser16, form interhelical hydrogen bonds in the\nclosed state but become separated by water molecules in the open state.\nThis is manifested by Asn15-Ser16 correlation signals at neutral pH\nand the loss of these correlations and the appearance of water cross\npeaks with Ser16 at acidic pH in the presence of Ca<sup>2+</sup>.\nFinally, the guanidinium side chain of the C-terminal Arg38 undergoes\nfast reorientations in the closed state but becomes more restricted\nin the open state. These results provide evidence for a dynamic and\nhydrogen-bonded N-terminal polar network that recruits and relays\nprotons and Ca<sup>2+</sup> in a lipid-dependent manner. Once inside,\nthe ions permeate past the hydrophobic middle of the transmembrane\ndomain with the help of enhanced hydrophilicity of the C-terminal\nchannel lumen due to the insertion of the Arg38 side chain into the\npore.