Structural and Functional Basis of SARS-CoV-2 Entry by Using Human ACE2

21 Pages·2020·4.957 MB·English

by  Qihui Wang, Yanfang Zhang, Lili Wu, Huan Zhou, Jinghua Yan, Jianxun Qi

Article
Structural and Functional Basis of SARS-CoV-2 Entry
by Using Human ACE2
Graphical Abstract Authors
QihuiWang,YanfangZhang,LiliWu,...,
HuanZhou,JinghuaYan,JianxunQi
Correspondence
[email protected](J.Y.),
[email protected](J.Q.)
In Brief
ThecrystalstructureoftheC-terminal
domainoftheSARS-CoV-2spikeprotein
incomplexwithhumanACE2reveals
insightsintothemechanismsofbinding
ofthisvirusanditsdifferences
fromSARS.
Highlights
d SARS-CoV-2interactswithhACE2viaSproteinCTD
d A2.5-A˚ structureofSARS-CoV-2-CTDincomplexwith
hACE2isresolved
d TheSARS-CoV-2-CTDdisplaysstrongeraffinityforhACE2
comparedwithSARS-RBD
d SARS-CoV-2-CTDisantigenicallydifferentfromSARS-RBD
Wangetal.,2020,Cell181,894–904
May14,2020ª2020ElsevierInc.
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https://doi.org/10.1016/j.cell.2020.03.045
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Article
Structural and Functional Basis
of SARS-CoV-2 Entry by Using Human ACE2
QihuiWang,1,2,3,15YanfangZhang,3,4,5,15LiliWu,1,4,15ShengNiu,3,6,15ChunliSong,1,7,15ZengyuanZhang,3,4
GuangwenLu,8ChengpengQiao,1YuHu,3,9Kwok-YungYuen,10,11QishengWang,12HuanZhou,12JinghuaYan,1,2,3,7,13,*
andJianxunQi3,14,16,*
1CASKeyLaboratoryofMicrobialPhysiologicalandMetabolicEngineering,InstituteofMicrobiology,ChineseAcademyofSciences,Beijing
100101,China
2ShenzhenKeyLaboratoryofPathogenandImmunity,ShenzhenThirdPeople’sHospital,Shenzhen518112,China
3CASKeyLaboratoryofPathogenicMicrobiologyandImmunology,InstituteofMicrobiology,ChineseAcademyofSciences,Beijing
100101,China
4UniversityoftheChineseAcademyofSciences,Beijing100049,China
5LaboratoryofProteinEngineeringandVaccines,TianjinInstituteofBiotechnology,Tianjin300308,China
6CollegeofAnimalScienceandVeterinaryMedicine,ShanxiAgriculturalUniversity,Taigu030801,China
7InstituteofPhysicalScienceandInformation,AnhuiUniversity,Hefei230039,China
8WestChinaHospitalEmergencyDepartment(WCHED),StateKeyLaboratoryofBiotherapyandCancerCenter,WestChinaHospital,
SichuanUniversity,andCollaborativeInnovationCenterofBiotherapy,Chengdu,Sichuan610041,China
9SchoolofLifeSciences,UniversityofScienceandTechnologyofChina,Hefei,Anhui230026,China
10StateKeyLaboratoryforEmergingInfectiousDiseases,TheUniversityofHongKong,Pokfulam,HongKongSpecialAdministrativeRegion
999077,China
11DepartmentofMicrobiology,TheUniversityofHongKong,Pokfulam,HongKongSpecialAdministrativeRegion999077,China
12ShanghaiSynchrotronRadiationFacility,ShanghaiAdvancedResearchInstitute,ChineseAcademyofSciences,Shanghai201204,China
13CollegeofLifeScience,UniversityoftheChineseAcademyofSciences,Beijing100049,China
14SavaidMedicalSchool,UniversityoftheChineseAcademyofSciences,Beijing100049,China
15Theseauthorscontributedequally
16LeadContact
*Correspondence:[email protected](J.Y.),[email protected](J.Q.)
https://doi.org/10.1016/j.cell.2020.03.045
SUMMARY
The recent emergence of a novel coronavirus (SARS-CoV-2) in China has caused significant public health
concerns.Recently,ACE2wasreportedasanentryreceptorforSARS-CoV-2.Inthisstudy,wepresentthe
crystalstructureoftheC-terminaldomain ofSARS-CoV-2(SARS-CoV-2-CTD)spike(S)protein incomplex
withhumanACE2(hACE2),whichrevealsahACE2-bindingmodesimilaroveralltothatobservedforSARS-
CoV.However,atomicdetailsatthebindinginterfacedemonstratethatkeyresiduesubstitutionsinSARS-
CoV-2-CTD slightly strengthen the interaction and lead to higher affinity for receptor binding than SARS-
RBD. Additionally, a panel of murine monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs)
againstSARS-CoV-S1/receptor-bindingdomain(RBD)wereunabletointeractwiththeSARS-CoV-2Spro-
tein, indicating notable differences in antigenicity between SARS-CoV and SARS-CoV-2. These findings
shed light on the viral pathogenesis and provide important structural information regarding development
of therapeutic countermeasures against the emerging virus.
INTRODUCTION (Wu et al., 2020; Zhou et al., 2020; Zhu et al., 2020). Later,
the International Committee on Taxonomy of Viruses (ICTV)
Emerging and re-emerging viruses are a significant threat to officially designated the virus SARS-CoV-2 (Coronaviridae
global public health (Gao, 2018). Since the end of 2019, Chi- Study Group of the International Committee on Taxonomy of
nese authorities have reported a cluster of human pneumonia Viruses, 2020), although many virologists argue that HCoV-19
casesinWuhanCity,China(Wangetal.,2020),andthedisease is more appropriate (Jiang et al., 2020). As of February 24,
was designated coronavirus disease 2019 (COVID-19). These 2020, 79,331 laboratory-confirmed cases have been reported
cases showed symptoms such as fever and dyspnea and to the WHO globally, with 77,262 cases in China, including
were diagnosed as viral pneumonia (Tan et al., 2020; Zhu 2,595deaths(https://www.who.int/).Inaddition,29othercoun-
et al., 2020). Whole-genome sequencing results showed that tries have confirmed imported cases of SARS-CoV-2 infection
the causative agent was a novel coronavirus that was initially (https://www.who.int/), raising great public health concerns
named 2019-nCoV by the World Health Organization (WHO) worldwide.
894 Cell181,894–904,May14,2020ª2020ElsevierInc.
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SARS-CoV-2 is the seventh coronavirus that is known to (Lai et al., 2007). S1 can be further divided into an N-terminal
causehumandisease.Coronaviruses(CoVs)areagroupoflarge domain (NTD) and a C-terminal domain (CTD), both of which
andenvelopedviruseswithpositive-sense,single-strandedRNA canfunctionasareceptor-bindingentity(e.g.,SARS-CoVand
genomes(Laietal.,2007;LuandLiu,2012).Thevirusescanbe MERS-CoV utilize the S1 CTD to recognize the receptor (also
classifiedintofourgenera:alpha,beta,gamma,anddeltaCoVs calledreceptorbindingdomain[RBD])(Lietal.,2005;Luetal.,
(Woo et al., 2009; https://talk.ictvonline.org/). Previously 2013), whereas mouse hepatitis CoV engages the receptor
identified human CoVs that cause human disease include the with its S1 NTD (Taguchi and Hirai-Yuki, 2012)). The region in
alphaCoVs hCoV-NL63 and hCoV-229E and the betaCoVs SARS-CoV-2SproteinthatisresponsibleforhACE2interaction
HCoV-OC43, HKU1, severe acute respiratory syndrome CoV remainsunknown.
(SARS-CoV), and Middle East respiratory syndrome CoV Inthisstudy,byutilizingimmunostainingandflowcytometryas-
(MERS-CoV)(Luetal.,2015;WeversandvanderHoek,2009). says,wefirstidentifytheS1CTD(SARS-CoV-2-CTD)asthekey
Both alphaCoVs and the betaCoVs HCoV-OC43 and HKU1 region in SARS-CoV-2 that interacts with the hACE2 receptor.
cause self-limiting common cold-like illnesses (Chiu et al., Wesubsequentlysolveda2.5-A˚ crystalstructureofSARS-CoV-
2005; Gorse et al., 2009; Jean et al., 2013; Jev(cid:1)snik et al., 2-CTDincomplexwithhACE2,whichrevealsareceptor-binding
2012).However,SARS-CoVandMERS-CoVinfectioncanresult mode similar overall to that observed for the SARS-CoV RBD
inlife-threateningdiseaseandhavepandemicpotential.During (SARS-RBD).However,SARS-CoV-2-CTDformsmoreatomicin-
2002–2003, SARS-CoV initially emerged in China and swiftly teractions with hACE2 than SARS-RBD, which correlates with
spread to other parts of the world, causing more than 8,000 datashowinghigheraffinityforreceptorbinding.Notably,apanel
infections and approximately 800 related deaths worldwide of monoclonal antibodies (mAbs) as well as murine polyclonal
(WHO, 2004). In 2012, MERS-CoV was first identified in the antisera against SARS-S1/RBD were unable to bind to the
MiddleEastandthenspreadtoothercountries(Ksiazeketal., SARS-CoV-2Sprotein,indicatingnotabledifferencesinantige-
2003;Zakietal.,2012).AsofNovember2019,atotalof2,494 nicity between SARS-CoV and SARS-CoV-2, suggesting that
MERS cases with 858 related deaths have been recorded in thepreviouslydevelopedSARS-RBD-basedvaccinecandidates
27 countries globally (https://www.who.int/emergencies/ areunlikelytobeofanyclinicalbenefitforSARS-CoV-2prophy-
mers-cov/en/). Notably, new cases of MERS-CoV infecting laxis.Takentogether,thesedatashedlightonviralentryandpath-
humans are still being reported (https://www.who.int/csr/don/ ogenesisandwillhopefullyinspirenewtargetedtreatmentsfor
archive/disease/coronavirus_infections/en/). SARS-CoV and thisemergingpathogen.
MERS-CoV are zoonotic pathogens originating from animals.
Detailed investigations indicate that SARS-CoV is transmitted RESULTS
from civet cats to humans and MERS-CoV from dromedary
camels to humans (Azhar et al., 2014; Ge et al., 2013; Guan SARS-CoV-2AppliestheCTDtoInteractwithhACE2
etal.,2003).ThesourceofSARS-CoV-2,however,isstillunder Throughbioinformaticsanalysis,theSARS-CoV-2Sproteinhas
investigationbutlinkedtoawetanimalmarket(Zhuetal.,2020; beenshowntodisplaycharacteristicCoVSfeatures,includinga
The2019-nCoVOutbreakJointFieldEpidemiologyInvestigation S1regioncontainingtheNTDandCTD,S2,atransmembranere-
TeamandLi,2020). gion,andashortcytoplasmicdomain(FigureS1A).Phylogenetic
Virus infections initiate with binding of viral particles to host studiesrevealthatSARS-CoV-2belongstoagroupcontaining
surfacecellularreceptors.Receptorrecognitionisthereforean SARS-CoVaswellastwobat-derivedSARS-likeviruses,ZC45
importantdeterminantofthecellandtissuetropismofavirus. andZCX21(FiguresS1B–S1D).Recently,hACE2wasreported
Inaddition,thegainoffunctionofavirustobindtothereceptor tobethereceptorofSARS-CoV-2(Zhouetal.,2020).Because
counterpartsinotherspeciesisalsoaprerequisiteforinter-spe- SARS-CoV utilizes its S1 CTD, otherwise known as the RBD,
cies transmission (Lu et al., 2015). Interestingly, with the to recognize the same receptor, we decided to test whether
exceptionofHCoV-OC43andHKU1,bothofwhichhavebeen the CTD in SARS-CoV-2 is also the key region for interaction
shown to engage sugars for cell attachment (Li et al., 2005), withitsreceptorhACE2.
theotherhumanCoVsrecognizeproteinaceouspeptidasesas WepreparedaseriesofmouseFc(mFc)-fusedSARS-CoV-2S
receptors. HCoV-229E binds to human aminopeptidase N protein preparations, including S1 (SARS-CoV-2-S1), the NTD
(hAPN) (Li et al., 2019), and MERS-CoV interacts with human (SARS-CoV-2-NTD),andtheCTD,andsubsequentlyvisualized
dipeptidyl peptidase 4 (hDPP4 or hCD26) (Lu et al., 2013; Raj theirbindingtoGFP-taggedhACE2expressedonthecellsur-
et al., 2013). Although they belong to different genera, face via confocal fluorescence microscopy. As a control, we
SARS-CoV and hCoV-NL63 interact with human angiotensin- also prepared the Fc fusion proteins for SARS-RBD and
converting enzyme 2 (hACE2) for virus entry (Hofmann et al., MERS-RBD and tested these in parallel with the SARS-CoV-2
2005; Li et al., 2003; Wu et al., 2009). After the outbreak of proteins.Asexpected,SARS-RBDshowedco-localizationwith
COVID-19,ChinesescientistspromptlydeterminedthatSARS- hACE2andMERS-RBDwithhCD26.ForthenovelCoVproteins,
CoV-2alsoutilizeshACE2forcellentry(Zhouetal.,2020). SARS-CoV-2-S1 and SARS-CoV-2-CTD co-localized with
In CoVs, the entry process is mediated by the envelope- hACE2onthecellsurface.TheSARS-CoV-2-NTDprotein,how-
embedded surface-located spike (S) glycoprotein (Lu et al., ever,wasincapableofbindingtohACE2.Inaddition,noneofthe
2015).ThisSproteinwould,inmostcases,becleavedbyhost SARS-CoV-2proteinsinteractedwithhCD26(Figure1).
proteases into the S1 and S2 subunits, which are responsible Wefurther tested binding oftheviralproteinsto cell-surface
for receptor recognition and membrane fusion, respectively hACE2 via flow cytometry. Consistently, SARS-CoV-2-S1 and
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Figure1. SARS-CoV-2-S1andSARS-CoV-2-CTDCo-localizewithhACE2
HEK293TcellsweretransfectedwithpEGFP-N1-hACE2(leftpanels,hACE2-GFP)orpEGFP-C1-hCD26(rightpanels,hCD26-GFP).Twenty-fourhourslater,the
cellswereincubatedwithsupernatantcontainingmFc-taggedSARS-CoV-2-S1(SARS-CoV-2-S1-mFc),SARS-CoV-2-NTD(SARS-CoV-2-NTD-mFc),SARS-
CoV-2-CTD(SARS-CoV-2-CTD-mFc),MERS-RBD(MERS-RBD-mFc),orSARS-RBD(SARS-RBD-mFc)proteinsandsubsequentlyincubatedwithanti-mouse
IgG(mIgG)antibodyconjugatedwithA594(anti-mIgG/A594).NucleiwerestainedwithDAPI.AllimageswereobtainedbyconfocalmicroscopyusingaLeicaSP8
(3100oilimmersionobjectivelens).Thescalebarineachpanelindicates8mm.Thedatashownarerepresentativeoftwoindependentexperiments.
SeealsoFigureS2.
SARS-CoV-2-CTD,butnotSARS-CoV-2-NTD,showedstrongaf- structural domains (Han et al., 2017). One is the conserved
finityforhACE2(FigureS2A).NoneofthenovelCoVproteinsinter- coresubdomainwithfiveantiparallelbstrandsandaconserved
actedwithhCD26orhAPN(FiguresS2BandS2C).Inaddition,sol- disulfide bond between bc2 and bc4 (Figures 2B and S1D).
ublehACE2,butnothCD26orhAPN,wasshowntoinhibitthe The other isthe external subdomain, which is dominated bya
interactionbetweenviralproteins,withcellsexpressinghACE2 disulfide bond-stabilized flexible loop that connects two small
inadose-dependentmanner(FiguresS2D–S2I).Takentogether, bstrands.ThecomplexstructuredatashowthatSARS-CoV-2-
theseresultsclearlydemonstratethatSARS-CoV-2iscapableof CTD utilizes its external subdomain to recognize subdomain I
binding,viatheviralCTD,tohACE2. inthehACE2NTD(Figure2A;Towleretal.,2004).
Further analysis was performed to identify key residues
ComplexStructurebetweenSARS-CoV-2andhACE2 involved incomplex formation. Aminoacidslocated withinthe
Wethenattemptedtostudythestructuralbasisofthevirus-re- van der Waals (vdw) contact distance (4.5-A˚-resolution cutoff)
ceptor interaction. Weprepared theSARS-CoV-2-CTD/hACE2 between the viral ligand and receptor were selected (Table 2),
complex by in vitro mixture of the two proteins and isolated andaseriesofhydrophilicresidueslocatedalongtheinterface
complexes via size exclusion chromatography. The complex werefoundtoformasolidnetworkofH-bondandsaltbridgein-
structure was solved to 2.5-A˚ resolution (Table 1), with one teractions (Figure 2). These strong polar contacts include the
SARS-CoV-2-CTD binding to a single hACE2 molecule in the SARS-CoV-2-CTDresidueA475interactingwithhACE2residue
asymmetric unit. For hACE2, clear electron densities could be S19,N487withQ24(Figures2CandS3A),E484withK31,and
tracedfor596residuesfromS19toA614oftheN-terminalpepti- Y453withH34(Figures2DandS3B).ResidueK417,locatedin
dasedomainaswellasglycansN-linkedtoresidues53,90,and helixa3oftheCTDcoresubdomain,wasshowntocontribute
322(Figure2A). ionicinteractionswithhACE2D30(Figures2DandS3B).Notably,
Inthecomplexstructure,theSARS-CoV-2-CTDcontains195 thebulgedloopsinSARS-CoV-2-CTD,thea1’/b1’loopandb20/
consecutivedensity-traceableresiduesspanningT333toP527 h1’loop,properlypositionseveralresidues(G446,Y449,G496,
togetherwithN-linkedglycosylationatN343.Similartootherre- Q498, T500, and G502) in close proximity with hACE2 amino
ported betaCoV CTD structures, this protein also exhibits two acidsD38,Y41,Q42,K353,andD355,formingaconcentration
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Ca atoms (Figure 3A). In comparison with the SARS-RBD, the
Table1. DataCollectionandRefinementStatistics
majorityofthesecondarystructureelementsarewellsuperim-
SARS-CoV-2-CTDandhACE2
posed in SARS-CoV-2-CTD, with the exception of the b10/b20
DataCollection
loop, whichshowedthemostsequencevariation betweenthe
Spacegroup P41212 twoligands(Figures3AandS1D).
CellDimensions TheoverallstructureoftheSARS-CoV-2-CTD/hACE2complex
a,b,c(A˚) 104.45,104.45,229.79 isverysimilartothepreviouslyreportedstructureofSARS-RBD
a,b,c((cid:2)) 90.00,90.00,90.00 bound to the same receptor with anRMSDof 0.431 A˚ for 669
Resolution(A˚) 50.00–2.50(2.59–2.50) equivalentCaatoms(Lietal.,2005;Figures3A–3C).Consistent
withthishighdegreeofsimilarity,thesolubleSARS-RBDblocks
Uniquereflections 44,981(43,84)
theinteractionbetweentheSARS-CoV-2ligandwithhACE2ina
Completeness(%) 100.0(100.0)
concentration-dependentmanner(FiguresS2JandS2K).Further
Rmerge 0.129(1.147) detailedcomparisonofthereceptorbindinginterfacebetweenthe
I/sI 26.7(3.3) twovirusesreveals that,amongthe24residuesinhACE2that
CC (%) 0.999(0.867) make vdw contacts with either CTD, 15 amino acids display
1/2
Redundancy 21.6(22.3) morecontactswiththeSARS-CoV-2-CTD(Table2).TheSARS-
CoV-2-CTD binding interface also has more residues than
Refinement
SARS-RBD(21versus17)thatdirectlyinteractwithhACE2,form-
Resolution(A˚) 34.50-2.50
ingmorevdwcontacts(288versus213)aswellasH-bonds(16
Numberofreflections 44861
versus11)(Tables2andS1).Consistently,SARS-CoV-2-CTDin
Rwork/Rfree 0.1846/0.2142 complex with hACE2 buries larger surface areas than SARS-
NumberofAtoms RBD(1773A˚2versus1686A˚2).
Protein 6,461 Notably,themostvariableloop(b1’/b20loop)contributessub-
Ligand/ion 1 stantiallymorevdwcontactsinSARS-CoV-2-CTDthanforthe
SARS-RBD(115versus53)(Figure3D;TableS1).Specifically,
Water 322
F486 in SARS-CoV-2, instead of I472 in SARS-RBD, forms
B-factors
strong aromatic-aromatic interactions with hACE2 Y83, and
Protein 44.1
E484 in the SARS-CoV-2-CTD, instead of P470 in the
Ligand/ion 38.3
SARS-RBD,formsionicinteractionswithK31(Figure3D).
Water 40.4
RMSDs TheInteractionbetweenSARS-CoV-2-CTDandhACE2
Bondlengths(A˚) 0.005 IsSpecificandDisplays4-FoldStrongerAffinity
Bondangles((cid:2)) 0.799 ComparedwiththeSARS-RBD
In light of the increased atomic interactions between hACE2
RamachandranStatistics(%)
with the SARS-CoV-2-CTD compared with the SARS-RBD,
Favored 98.60
we speculated that the former should bind to the receptor
Allowed 1.02
with stronger affinity than the latter. To test this hypothesis,
Disallowed 0.38
we performed real-time surface plasmon resonance (SPR) as-
Valuesinparenthesesareforthehighest-resolutionshell. says. The mFc-tagged S-domain proteins were captured by
anti-mouse IgG (mIgG) antibodies that were immobilized on
the chip and tested for binding with gradient concentrations
ofH-bonds(Figures2EandS3C).Furthervirus-receptorcontacts of the soluble ectodomain proteins of hACE2 and hCD26. As
include SARS-CoV-2-CTD Y489 and F486 packing against assay controls, SARS-RBD and MERS-RBD were found to
hACE2residuesF28,L79,M82,andY83,formingasmallpatch readily interact with their respective canonical receptors (Fig-
of hydrophobic interactions at the interface (Figures 2C and ures 4A and 4D). SARS-CoV-2-S1 and SARS-CoV-2-CTD
S3A). Overall, the virus-receptor engagement is dominated by bound to hACE2 but not to hCD26 (Figures 4E, 4F, 4I, and
polarcontactsmediatedbythehydrophilicresidues.Insupport 4J). The recorded binding profiles revealed typical slow-on/
of this hypothesis, a single K353A mutation was sufficient to slow-off kinetics, as observed with the SARS-CoV and
abolishtheseinteractions(FigureS2L). MERS-CoV proteins. The equilibrium dissociation constants
(K ) of SARS-CoV-2-S1 and SARS-CoV-2-CTD binding to
D
ComparisonoftheBindingInterfacesbetweenhACE2/ hACE2 were calculated to be 94.6 ± 6.5 nM and 133.3 ±
SARS-CoV-2-CTDandhACE2/SARS-RBD 5.6 nM, respectively. These values represent ~4-fold higher
SARS-CoV-2-CTDexhibitssignificantstructuralhomologytoits binding affinities than that observed for the SARS-RBD
SARS-CoVhomolog,inagreementwithhighsequenceidentity engaging the same receptor, which was determined to be
betweenthetwomolecules(~73.9%)(FigureS1C).Superimpo- 408.7 ± 11.1 nM (Figure 4). Taken together, the increased
sition of the SARS-CoV-2-CTD structure onto a previously atomic interactions between the hACE2 and SARS-CoV-2-
reported SARS-RBD structure (PDB: 2GHV) revealed a root- CTD binding region leads to ~4-fold higher binding affinity
mean-square deviation (RMSD) of 0.475 A˚ for 128 equivalent comparedwiththe SARS-RBD.
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Figure2. TheComplexStructureofSARS-CoV-2-CTDBoundtohACE2
(A)Acartoonrepresentationofthecomplexstructure.ThecoresubdomainandexternalsubdomaininSARS-CoV-2-CTDarecoloredcyanandorange,
respectively.hACE2subdomainIandIIarecoloredvioletandgreen,respectively.Therightpanelwasobtainedbyanticlockwiserotationoftheleftpanelalonga
longitudinalaxis.Thecontactingsitesarefurtherdelineatedin(C)–(E)fortheaminoacidinteractiondetails.
(B)AcartonrepresentationoftheSARS-CoV-2-CTDstructure.Thesecondarystructuralelementsarelabeledaccordingtotheiroccurrenceinsequenceand
locationinthesubdomains.Specifically,thebstrandsconstitutingthecoresubdomainarelabeledwithanextrac,whereastheelementsintheexternalsub-
domainarelabeledwithanextraprimesymbol.ThedisulfidebondsandN-glycanlinkedtoN343areshownassticksandspheres,respectively.
(C–E)Keycontactsitesaremarkedwiththeleft,middleandrightboxin(A)andfurtherdelineatedforinteractiondetails,respectively.Theresiduesinvolvedare
shownandlabeled.
SeealsoFiguresS1,S2,andS3andTableS1.
SARS-CoV-2ExhibitsDistinctEpitopeFeaturesinthe distinctstructuralcharacteristicsintheRBDexternalsubdomain
RBDfromSARS-CoV thatmediatesreceptorbinding(Lietal.,2005;Luetal.,2013).In
To conclude, we set out to investigate the epitope features of the positive control, anti-SARS-RBD antibodies, but not anti-
SARS-CoV-2SbyusingapanelofmurinemAbsdirectedagainst MERS-RBD antibodies, potently bound to cells expressing
SARS-CoV S, including the B30A38, A50A1A1, and C31A12 SARS-CoV S, as expected (Figures 5C and 5D). Nonetheless,
antibodies, which recognize SARS-CoV S1, and mAbs 1–3, neitheroftheantibodypreparationsboundtoSARS-CoV-2S(Fig-
whichrecognizetheSARS-RBD(FigureS1D;Wenetal.,2004; ures 5E and 5F). In agreement with this observation, although
Zhang et al., 2009). Using flow cytometry, all six mAbs were SARS-CoV-2-CTDisstructurallysimilartotheSARS-RBDstruc-
observed to effectively bind to cells expressing SARS-CoV S. tures(Figure3),theelectrostaticsurfacepotentialmapsofthese
NoneofthemAbs,however,interactedwithSARS-CoV-2S(Fig- proteinsweredifferent(Figure5Gand5H),whichmightexplain
ures5Aand5B). thedifferingimmunogenicityofthetwoligands.Therefore,there-
IncomparisonwithalimitednumberofmAbs,polyclonalanti- sultshighlightdistinctepitopefeaturesbetweenSARS-RBDand
bodiesprovideamorecomprehensiveviewonpotentialepitope SARS-CoV-2-CTD,althoughbothcanengagehACE2.
differences. In light of the determinant role of SARS-RBD and
MERS-RBD in receptor recognition (Li et al., 2003, 2005; Lu DISCUSSION
et al., 2013; Raj et al., 2013), the majority of neutralizing
antibodieshavebeenshowntotargettheRBD,exertingneutral- TherecentemergenceofSARS-CoV-2infectioninChinahasled
izationactivitybydisruptingvirus/receptorengagement(Duetal., tomajorpublichealthconcerns.ACE2hasbeenreportedtobe
2009;Wangetal.,2016).Wethereforefurtherpreparedmurine thereceptorforthisnovelCoV(Hoffmannetal.,2020;Zhouetal.,
polyclonalantibodiesagainstSARS-RBDandMERS-RBD.These 2020).Inthisstudy,wedeterminedthekeyregioninSARS-CoV-
twoviralRBDsshareverylimitedsequenceidentityandexhibit 2 that is responsible for the interaction with the receptor and
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toutilizethehACE2receptorforcellentry.Thecomplexstruc-
Table2. ComparisonofhACE2BindingtoSARS-CoV-2-CTDand
SARS-RBD tures of hCoV-NL63 CTD and SARS-RBD bound to hACE2
havebeenreportedpreviously(Lietal.,2005;Wuetal.,2009).
hACE2 SARS-CoV-2-CTD SARS-RBD
Although hCoV-NL63 CTD and SARS-RBD are structurally
S19(7/1) A475(3,1),G476(4) P462(1)
distinct, the two viral ligands recognize and engage sterically
Q24(24/6) A475(4),G476(5), N473(6,1)
overlappingsitesinthereceptor(Li,2015).Thecomplexstruc-
N487(15,1)
tureofSARS-CoV-2-CTDtogetherwithhACE2revealsthatthe
T27(15/8) F456(5),Y473(1), L443(3),Y475(5)
majorityofbindingsitesofSARS-CoV-2inhACE2alsooverlap
A475(2),Y489(7)
theSARS-CoVbindingsite.Theobservationsfavorascenario
F28(7/7) Y489(7) Y475(7) wheretheseCoVshaveevolvedtorecognizea‘‘hotspot’’region
D30(10/2) K417(4,1),L455(2), Y442(2) inhACE2forreceptorbinding.
F456(4) During the revision of our manuscript, the full-length hACE2
K31(19/12) L455(2),F456(5), Y442(6),Y475(6) structure was reported to form a dimer in the presence of
E484(1),Y489(6), B0AT1(anaminoacidtransporter),asrevealedbycryoelectron
F490(2),Q493(3)
microscopy(cryo-EM)analysis(Yanetal.,2020).Theyalsore-
H34(20/10) Y453(5,1),L455(9), Y440(5,1), ported the cryo-EM structure of dimeric hACE2-B0AT1 bound
Q493(6) Y442(1),N479(4) to two SARS-CoV-2-CTDs, with each molecule bound to an
E35(8/0) Q493(8) – hACE2monomer,withalocalresolutionof3.5A˚ attheinterface.
E37(7/4) Y505(7) Y491(4) OurcrystalstructureofSARS-CoV-2-CTD/hACE2iswellsuper-
D38(15/11) Y449(9,1),G496(5), Y436(9,2), imposedwiththecryo-EMstructure,withanRMSDof1.019A˚
Q498(1) G482(1),Y484(1) over 722 pairs of Ca atoms. Notably, two cryo-EM structures
Y41(23/25) Q498(8),T500(7,1), Y484(9),T486(8,1), oftrimericSARS-CoV-2Sproteinswerealsopublishedrecently,
N501(8,1) T487(8) withthereceptorbindingregionburiedorexposed(Wallsetal.,
Q42(16/9) G446(4,1),Y449(4,1), Y436(5,1),Y484(4) 2020;Wrappetal.,2020),whichisconsistentwiththestructural
Q498(8,3) featuresofMERS-CoVandSARS-CoVSproteins(Yuanetal.,
L45(4/3) Q498(3),T500(1) Y484(2),T486(1) 2017).Furtherstructurealignmentsshowthatthecrystalstruc-
ture of SARS-CoV-2-CTD in the complex also fits well with its
L79(2/2) F486(2) L472(2)
counterpartsinthecryo-EMstructures,withRMSDsof0.724A˚
M82(9/4) F486(9) L472(4)
(exposed state) and 0.742 A˚ (buried states) related to PDB:
Y83(20/10) F486(11),N487(8,1), N473(8,2),Y475(2) 6VSB and 0.632 A˚ (exposed state) and 0.622 A˚ (buried state)
Y489(1)
relatedtoPDB:6VYB,respectively.Theseresultsindicatethat
Q325(0/4) – R426(2),I489(2)
the crystal structure of the complex is consistent with the
E329(0/6) – R426(6,1) respectivecryo-EMstructuresandprovidemoredetailedbind-
N330(8/11) T500(8) T486(11,1) inginformation.
K353(50/48) G496(7,1),N501(11), Y481(1),G482(3), ConsideringthehighsequenceidentitybetweenSARS-CoV-
G502(4,1),Y505(28) Y484(2),T487(11), 2-CTD and SARS-RBD, atomic comparisons of the two viral
G488(6,1),Y491(25) ligandsbindingthesamereceptorwereperformed.Atomicde-
G354(11/10) Y502(7),Y505(4) G488(7),Y491(3) tailsrevealmoreinteractionsinSARS-CoV-2-CTD/hACE2than
D355(9/15) T500(8,1),G502(1) T486(8),T487(3), inSARS-RBD/hACE2,includingmoreengagedresidues,more
G488(4) vdw contacts, more H-bonds, as well as larger buried surface
R357(3/4) T500(3) T486(4) areas.Interestingly,theb1’/b20loop,whichisthemostvariable
R393(1/1) Y505(1) Y491(1) region between SARS-CoV-2-CTD and SARS-RBD, confers
moreinteractionstoSARS-CoV-2-CTD/hACE2,includingstrong
Total 288(16) 213(11)
interactions, such as aromatic-aromatic interactions and ionic
ThenumbersinparenthesesofhACE2residuesrepresentthenumberof
interactions, in contrast to the SARS-RBD b1’/b20 loop. A
vdwcontactsbetweentheindicatedresiduewithSARS-CoV-2-CTD(the
recentlypublishedpaperalsoindicatesthattheSARS-CoV-2S
former)andSARS-RBD(thelatter).Thenumbersinparenthesesofeither
ligandresidues representthenumbersofwdwcontactstheindicated proteinbindshACE2withhigheraffinitythantheSARS-CoVS
residuesconferred.Thenumberswithunderlinesuggestnumbersofpo- protein (Wrapp et al., 2020), which was shown in this report
tential H-bonds between the pairs of residues. wdw contact was aswell.
analyzedatacutoffof4.5A˚ andH-bondsatacutoffof3.5A˚.Seealso ProteolysisoftheSproteinintoS1andS2isanotherprereq-
TableS1. uisite for CoVs infection. MERS-Uganda and the bat CoV
HKU4canreadilyinteractwithhCD26,buttheybothrequirepro-
teaseactivationforcellentry(Kametal.,2009;Matsuyamaand
solved the crystal structure of SARS-CoV-2-CTD in complex Taguchi,2009;Menacheryetal.,2015,2020;Wangetal.,2014).
withhACE2. Arecentstudyshowsthat,incontrastwithSARS-CoVS,which
ConsideringthenewlyidentifiedSARS-CoV-2,atotalofseven does not contain furin recognition sites between S1 and S2,
humanCoVshavebeenreportedsofar.Oftheseviruses,three SARS-CoV-2Scontainsonepotentialcleavagesiteandcould
(hCoV-NL63, SARS-CoV, and SARS-CoV-2) have been shown beefficientlyprocessedintoS1andS2(Hoffmannetal.,2020).
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Figure3. ComparisonoftheSARS-CoV-2-CTD/hACE2andSARS-RBD/hACE2BindingSites
(A)OverallsimilarreceptorbindingmodeswereobservedforSARS-CoV-2-CTDandSARS-RBD.SuperimpositionofthestructureofSARS-CoV-2-CTD(external
subdomaininorangeandcoresubdomainincyan)boundtohACE2(violet)andacomplexstructureofSARS-RBD(ingray)withhACE2(yellow)areshown.The
loopexhibitingvariantconformationsishighlightedbyadashedoval.
(B)hACE2displayedinsurfaceview.ResiduesthatinteractwiththeSARS-CoV-2-CTDaremarked.
(C)hACE2displayedinsurfaceview.ResiduesthatinteractwiththeSARS-RBDaremarked.
(D)ResiduessubstitutionsinSARS-CoV-2-CTDslightlystrengthentheinteractionwiththereceptorcomparedtotheSARS-RBD.Theaminoacidsequencesof
theloopspecifiedin(A)werealignedbetweentheSARS-CoV-2-CTDandtheSARS-RBD.Thenumbersshowthevdwcontactsbetweenthereceptorwiththe
indicatedSARS-CoV-2-CTDresidues(abovethesequence)orSARS-RBDresidues(belowthesequence).Numbersinparenthesesindicatethenumberof
potentialH-bondsconferredbytheindicatedresidues.Theredandbluearrowsrepresenttheaminoacidsthatformionicandaromatic-aromaticinteractionswith
thereceptor,respectively.
SeealsoFigureS1andTableS1.
TheserineproteaseTMPRSS2hasbeenreportedtocontribute teins have been shown to efficiently induce production of
toprimingoftheSARS-CoV-2Sprotein,andaTMPRSS2inhib- neutralizing antibodies (Du et al., 2009; Wang et al., 2016).
itorapprovedforclinicalusewasabletoblockentry.Theauthors However, because of the observed differences in antigenicity
postulated that the TMPRSS2 inhibitor might be a treatment and electrostatic distribution between SARS-CoV and SARS-
option(Hoffmannetal.,2020). CoV-2,itisunclearwhetherpreviouslydevelopedSARS-RBD-
AlthoughSARS-CoVandSARS-CoV-2sharemorethan70% basedvaccinecandidates,suchassubunitvaccines,willconfer
sequenceidentityintheSprotein,andbothengagehACE2via effective SARS-CoV-2 prophylaxis. During the revision of our
the CTD, we find that the two viruses CTDs are antigenically manuscript,otherstudieshavereportedthatSARS-CoVS-eli-
distinct.WhenusingapanelofmAbstargetingSARS-CoVS1/ citedpolyclonalantibodiesinmiceandpatientspotentlyneutral-
CTD, none of the antibodies were able to recognize SARS- izedSARS-CoV-2S-mediatedentryintocells(Hoffmannetal.,
CoV-2 S. mAb1 and mAb2/mAb3 used in the above assay 2020; Walls et al., 2020). Notably, the S2 regions between
havebeendeterminedtobindtoSARS-CoVSprotein330–350 SARS-CoV and SARS-CoV-2 exhibit higher sequence identity
and 380–399, respectively (Zhang et al., 2009). However, the (~90%) and also contain neutralizing epitopes (Duan et al.,
bindingsitesfortheotherthreemAbs(B30A38,A50A1A1,and 2005;Wangetal.,2015).Thus,theefficacyofSARS-CoVvac-
C31A12), which were generated using SARS-CoV S1 as the cinestargetingSproteinsforSARS-CoV-2prophylaxisrequires
immunogen,remainelusive.Consistently,arecentlypublished furtherevaluationandstudy.
paper also reported similar results showing that three SARS- Inconclusion,CoVsarezoonoticpathogensandinfecthumans
RBD-directed mAbs, S230, m396, and 80R, were unable to viainter-speciestransmission.SARS-CoVandMERS-CoVaretwo
bind to SARS-CoV-2 (Wrapp et al., 2020). Furthermore, we notoriousexamplesofCoVscrossingthespeciesbarrierandre-
also demonstrate that polyclonal antisera directed against sultinginhumaninfection.Previousstudieshaveshownthatthe
SARS-RBD do not recognize the S protein of SARS-CoV-2. A twovirusesfirstjumpedfromtheirnaturalhosts(bats)toaninter-
comparison of the two viral ligands shows that they display mediate adaptive animal (e.g., dromedary camels for MERS-
divergentelectrostaticpotential,whichlikelyresultsindiffering CoV) before infecting humans (Azhar et al., 2014; Wang et al.,
immunogenicity despite both ligands showing a similar pro- 2014).Delineatingthiscross-speciestransmissionroutecouldbe
teinfold. highly instructive for disease control. Nevertheless, the natural
ConsideringthekeyroleoftheCTDinreceptorbinding,this hostandtheintermediateadaptiveanimal,ifany,forSARS-CoV-
receptorengagemententityisanidealimmunogenforvaccine 2remainsunknown.ThestructuralinformationforSARS-CoV-2-
development. For instance, SARS-RBD and MERS-RBD pro- CTDandhACE2showninthisstudyshouldshedlightontheviral
900 Cell181,894–904,May14,2020
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Figure4. SpecificInteractionsbetweenSARS-CoV-2-S1andSARS-CoV-2-CTDwithhACE2,CharacterizedbySPR
TheindicatedmFc-taggedproteinsinthesupernatantwerecapturedbyanti-mIgGantibodiesthatwereimmobilizedonthechipandsubsequentlytestedfor
bindingwithgradientconcentrationsofhACE2orhCD26,withthefollowingbindingprofilesshown.
(A)SARS-RBDbindingtohACE2.
(B)SARS-RBDbindingtohCD26.
(C)MERS-RBDbindingtohACE2.
(D)MERS-RBDbindingtohCD26.
(E)SARS-CoV-2-S1bindingtohACE2.
(F)SARS-CoV-2-S1bindingtohCD26.
(G)SARS-CoV-2-NTDbindingtohACE2.
(H)SARS-CoV-2-NTDbindingtohCD26.
(I)SARS-CoV-2-CTDbindingtohACE2.
(J)SARS-CoV-2-CTDbindingtohCD26.
(K)CulturesupernatantofHEK293Tcellswithouttransfection(NC)bindingtohACE2.
(L)CulturesupernatantofHEK293Tcellswithouttransfection(NC)bindingtohCD26.
Thevaluesshownarethemean±SDofthreeindependentexperiments.
inter-speciestransmissionroutebycharacterizingtheinteractions B SPRanalysis
betweenSandhACE2ofdifferentspeciesinthefuture. B Indirect immunofluorescence analysis and confocal
microscopy
STAR+METHODS B Immunizationofmice
B Crystallization
Detailedmethodsareprovidedintheonlineversionofthispaper B Datacollectionandstructuredetermination
andincludethefollowing: B Sequencesusedinthealignments
d QUANTIFICATIONANDSTATISTICALANALYSIS
d KEYRESOURCESTABLE B Bindingstudies
d LEADCONTACTANDMATERIALSAVAILABILITY B Flowcytometryanalysis
d EXPERIMENTALMODELANDSUBJECTDETAILS d DATAANDCODEAVAILABILITY
B Cells
d METHODDETAILS
SUPPLEMENTALINFORMATION
B Genecloning
B Proteinexpressionandpurification Supplemental Information can be found online at https://doi.org/10.1016/j.
B Flowcytometry cell.2020.03.045.
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Figure5. DifferentAntigenicitybetweentheSARS-CoV-2SandSARS-CoVSProteins
(AandB)HEK293TcellsweretransfectedwithpCAGGSplasmidscontainingFlag-taggedSARS-CoVS(A)orSARS-CoV-2S(B).Theindicatedpurifiedmurine
mAbsweresubsequentlyaddedtothetransfectedcellsbeforetheywerefixed,permeabilized,andstainedwithanti-Flag/fluoresceinisothiocyanate(FITC).
(CandD)HEK293TcellsweretransfectedwithpCAGGSplasmidsexpressingFlag-taggedSARS-CoVS.ThemurinepolyclonalseraagainstSARS-RBD(C)or
MERS-RBD(D)weresubsequentlyaddedtothetransfectedcellsbeforetheywerefixed,permeabilized,andstainedwithanti-Flag/FITC.
(EandF)HEK293TcellsweretransfectedwithpCAGGSplasmidsexpressingFlag-taggedSARS-CoV-2S.ThemurinepolyclonalseraagainstSARS-RBD(E)or
MERS-RBD(F)weresubsequentlyaddedtothetransfectedcellsbeforetheywerefixed,permeabilized,andstainedwithanti-Flag/FITC.
(G)ElectrostaticsurfaceviewofSARS-CoV-2-CTD.Thefirstpanelrepresentsthetopview.Theothersareyieldedbyrotationoftheformerpanelalonga
horizontalaxis.
(H)ElectrostaticsurfaceviewofSARS-RBD.Thefirstpanelrepresentsthetopview.Theothersareyieldedbyrotationoftheformerpanelalongahorizontalaxis.
ACKNOWLEDGMENTS XDB29040302), the National Key Research and Development Program of
China(2016YFD0500305),theNationalNaturalScienceFoundationofChina
WearegratefultoW.N.D.Gaoforrevisingthemanuscript.WethankY.Chen (81922044,81673358and81973228),andtheZhejiangUniversitySpecialSci-
andZ.Yang(InstituteofBiophysics,ChineseAcademyofSciences[CAS])for entificResearchFundforCOVID-19PreventionandControl(2020XGZX031).
technicalsupportfortheSPRanalysis.WethankX.Zhang(InstituteofMicro- Q.W. is supported by Youth Innovation Promotion Association CAS grant
biology,CAS)forhelpwithperformingconfocalimaging.WearegratefultoL. 2018119.G.L.issupportedbythespecialresearchfundonCOVID-19ofSi-
Dai(BeijingInstituteofLifeSciences,CAS)andK.Wen(SouthernMedicalUni- chuanProvince(2020YFS0010)andthespecialresearchfundonCOVID-19
versity) for kindly providing the mAbs/pAbs against SARS-CoV. We thank ofWestChinaHospitalSichuanUniversity(HX-2019-nCoV-004).J.Yissup-
L.Wangforhelpwiththephylogeneticsanalysis.Thisworkwassupported ported by Foundation of the NSFC Innovative Research Group grant
by the Strategic Priority Research Program of CAS (XDB29010202 and 81621091.
902 Cell181,894–904,May14,2020