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bioRxiv · 10.1101/2020.10.22.349522

Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites

Marquez-Miranda, V.·Rojas, M.·Duarte, Y.·Diaz-Franulic, I.·Holmgren, M.·Cachau, R.·Gonzalez-Nilo, F. D.
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Abstract

SARS-CoV-2 ORF3a is believed to form ion channels, which may be involved in the modulation of virus release, and has been implicated in various cellular processes like the up-regulation of fibrinogen expression in lung epithelial cells, downregulation of type 1 interferon receptor, caspase-dependent apoptosis, and increasing IFNAR1 ubiquitination. ORF3a assemblies as homotetramers, which are stabilized by residue C133. A recent cryoEM structure of a homodimeric complex of ORF3a has been released. A lower-resolution cryoEM map of the tetramer suggests two dimers form it, arranged side by side. The dimers cryoEM structure revealed that each protomer contains three transmembrane helices arranged in a clockwise configuration forming a six helices transmembrane domain. This domains potential permeation pathway has six constrictions narrowing to about 1 [A] in radius, suggesting the structure solved is in a closed or inactivated state. At the cytosol end, the permeation pathway encounters a large and polar cavity formed by multiple beta strands from both protomers, which opens to the cytosolic milieu. We modeled the tetramer following the arrangement suggested by the low-resolution tetramer cryoEM map. Molecular dynamics simulations of the tetramer embedded in a membrane and solvated with 0.5 M of KCl were performed. Our simulations show the cytosolic cavity is quickly populated by both K+ and Cl-, yet with different dynamics. K+ ions moved relatively free inside the cavity without forming proper coordination sites. In contrast, Cl- ions enter the cavity, and three of them can become stably coordinated near the intracellular entrance of the potential permeation pathway by an inter-subunit network of positively charged amino acids. Consequently, the central cavitys electrostatic potential changed from being entirely positive at the beginning of the simulation to more electronegative at the end.

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Marquez-Miranda, V., Rojas, M., Duarte, Y., Diaz-Franulic, I., Holmgren, M., Cachau, R., Gonzalez-Nilo, F. D.. 2020-10-22. Analysis of SARS-CoV-2 ORF3a structure reveals chloride binding sites. https://doi.org/10.1101/2020.10.22.349522

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