Abstract

SARS-CoV-2ORF3aisbelievedtoformionchannels,whichmaybeinvolvedinthemodulationofvirusrelease,andhasbeenimplicatedinvariouscellularprocessesliketheup-regulationoffibrinogenexpressioninlungepithelialcells,downregulationoftype1interferonreceptor,caspase-dependentapoptosis,andincreasingIFNAR1ubiquitination.ORF3aassembliesashomotetramers,whicharestabilizedbyresidueC133.ArecentcryoEMstructureofahomodimericcomplexofORF3ahasbeenreleased.Alower-resolutioncryoEMmapofthetetramersuggeststwodimersformit,arrangedsidebyside.Thedimer'scryoEMstructurerevealedthateachprotomercontainsthreetransmembranehelicesarrangedinaclockwiseconfigurationformingasixhelicestransmembranedomain.Thisdomain'spotentialpermeationpathwayhassixconstrictionsnarrowingtoabout1Åinradius,suggestingthestructuresolvedisinaclosedorinactivatedstate.Atthecytosolend,thepermeationpathwayencountersalargeandpolarcavityformedbymultiplebetastrandsfrombothprotomers,whichopenstothecytosolicmilieu.Wemodeledthetetramerfollowingthearrangementsuggestedbythelow-resolutiontetramercryoEMmap.Moleculardynamicssimulationsofthetetramerembeddedinamembraneandsolvatedwith0.5MofKClwereperformed.OursimulationsshowthecytosoliccavityisquicklypopulatedbybothK+andCl-,yetwithdifferentdynamics.K+ionsmovedrelativelyfreeinsidethecavitywithoutformingpropercoordinationsites.Incontrast,Cl-ionsenterthecavity,andthreeofthemcanbecomestablycoordinatedneartheintracellularentranceofthepotentialpermeationpathwaybyaninter-subunitnetworkofpositivelychargedaminoacids.Consequently,thecentralcavity'selectrostaticpotentialchangedfrombeingentirelypositiveatthebeginningofthesimulationtomoreelectronegativeattheend