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- PDB-9k6z: SARS-CoV-2 related bat coronavirus BANAL-52 spike in the locked state -

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Entry
Database: PDB / ID: 9k6z
TitleSARS-CoV-2 related bat coronavirus BANAL-52 spike in the locked state
ComponentsSpike glycoprotein
KeywordsVIRAL PROTEIN / SARS-CoV-2 related bat coronavirus BANA-52 spike with linoleic acid binding
Function / homology
Function and homology information


host cell endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / host cell plasma membrane / virion membrane ...host cell endoplasmic reticulum-Golgi intermediate compartment membrane / positive regulation of viral entry into host cell / receptor-mediated virion attachment to host cell / host cell surface receptor binding / endocytosis involved in viral entry into host cell / fusion of virus membrane with host plasma membrane / fusion of virus membrane with host endosome membrane / viral envelope / host cell plasma membrane / virion membrane / identical protein binding / membrane
Similarity search - Function
Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal ...Spike (S) protein S1 subunit, receptor-binding domain, SARS-CoV-2 / Spike (S) protein S1 subunit, N-terminal domain, SARS-CoV-like / Coronavirus spike glycoprotein S1, C-terminal / Coronavirus spike glycoprotein S1, C-terminal / Spike glycoprotein, N-terminal domain superfamily / Spike S1 subunit, receptor binding domain superfamily, betacoronavirus / Spike glycoprotein, betacoronavirus / Betacoronavirus spike (S) glycoprotein S1 subunit N-terminal (NTD) domain profile. / Spike glycoprotein S1, N-terminal domain, betacoronavirus-like / Betacoronavirus-like spike glycoprotein S1, N-terminal / Betacoronavirus spike (S) glycoprotein S1 subunit C-terminal (CTD) domain profile. / Spike (S) protein S1 subunit, receptor-binding domain, betacoronavirus / Betacoronavirus spike glycoprotein S1, receptor binding / Spike glycoprotein S2 superfamily, coronavirus / Spike glycoprotein S2, coronavirus, heptad repeat 1 / Spike glycoprotein S2, coronavirus, heptad repeat 2 / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 1 (HR1) region profile. / Coronavirus spike (S) glycoprotein S2 subunit heptad repeat 2 (HR2) region profile. / Spike glycoprotein S2, coronavirus / Coronavirus spike glycoprotein S2
Similarity search - Domain/homology
LINOLEIC ACID / Spike glycoprotein
Similarity search - Component
Biological speciesHorseshoe bat sarbecovirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.51 Å
AuthorsLi, Q.Q. / Cai, X. / Li, X.N. / Zhang, Y.B. / Li, R. / Kang, Z.R. / Wan, D.D. / Wang, J.X. / Yang, J.X. / Shi, J.X. ...Li, Q.Q. / Cai, X. / Li, X.N. / Zhang, Y.B. / Li, R. / Kang, Z.R. / Wan, D.D. / Wang, J.X. / Yang, J.X. / Shi, J.X. / Jin, S.L. / Peng, Y. / Zang, N. / Xie, Z.K. / Wan, Y.S. / Shang, J.
Funding support China, 3items
OrganizationGrant numberCountry
Other government32200113 China
Other government232301420013 China
Other government232102311001 China
CitationJournal: J Virol / Year: 2025
Title: Structural and functional constraints on spike activation and host protease utilization limit cell entry of SARS-CoV-2-related bat coronaviruses.
Authors: Qingqing Li / Xiao Cai / Xiaoning Li / Yibing Zhang / Ru Li / Zirui Kang / Didi Wan / Jiaxu Wang / Lili Li / Junxia Yang / Jianxiang Shi / Shuiling Jin / Xiangdong Sun / Ying Peng / Na Zang ...Authors: Qingqing Li / Xiao Cai / Xiaoning Li / Yibing Zhang / Ru Li / Zirui Kang / Didi Wan / Jiaxu Wang / Lili Li / Junxia Yang / Jianxiang Shi / Shuiling Jin / Xiangdong Sun / Ying Peng / Na Zang / Zhengkun Xie / Yushun Wan / Jian Shang /
Abstract: The persistent threat posed by SARS-CoV-2-related coronaviruses (SC2r-CoVs) in wildlife highlights the risk of zoonotic transmission. Cross-species infectivity is predominantly determined by spike (S) ...The persistent threat posed by SARS-CoV-2-related coronaviruses (SC2r-CoVs) in wildlife highlights the risk of zoonotic transmission. Cross-species infectivity is predominantly determined by spike (S) character and S-mediated cell entry. In this study, we systematically investigated BANAL-52 and BANAL-103, which exhibit the closest genetic proximity to SARS-CoV-2, focusing on their spike structures and functional characteristics. First, despite comparable receptor-binding domain (RBD)-ACE2 interactions, the spikes of BANAL-52 and BANAL-103 displayed significantly reduced ACE2 binding compared to SARS-CoV-2, suggesting impaired S activation. Second, Cryo-EM structural analyses revealed that BANAL-52 S is stabilized in a "locked" state through linoleic acid (LA) binding and an additional N370 glycan, whereas BANAL-103 S adopts a "closed" conformation due to a unique glycan network. Site-directed mutagenesis targeting the LA binding pocket confirmed that Y365 is related to S conformational transitions and viral entry. Third, both BANAL spikes relied predominantly on lysosomal proteases (e.g., cathepsins) for membrane fusion, unlike SARS-CoV-2, which utilizes a broader range of proteases (e.g., TMPRSS2 and furin). The introduction of a furin cleavage site enhanced the fusogenicity of BANAL spikes. Finally, sera from individuals who have recovered from SARS-CoV-2 effectively neutralized BANAL pseudoviruses, underscoring conserved antigenicity. Our findings elucidate structural and proteolytic barriers that restrict the zoonotic potential of these viruses and propose targeted surveillance strategies to preempt the emergence of SC2r-CoVs.
IMPORTANCE: The viral entry mechanisms, which are primarily related to the spike character, play a critical role in determining zoonotic potential. Among the currently identified SC2r-CoVs, BANAL-52 ...IMPORTANCE: The viral entry mechanisms, which are primarily related to the spike character, play a critical role in determining zoonotic potential. Among the currently identified SC2r-CoVs, BANAL-52 and BANAL-103 exhibit spike proteins with the highest sequence similarity to SARS-CoV-2, rendering them optimal models for comparative studies on S-mediated cell entry and cross-species transmission. In this study, we systematically investigated the molecular constraints governing the functionality of BANAL spikes, with a focus on S-ACE2 interactions, S activation, S structures, and host protease utilization. Notably, we resolved the cryo-EM structure of BANAL-52 S at neutral pH and the first cryo-EM structure of BANAL-103 S, revealing distinct glycan- and lipid-mediated stabilization of inactive states. Furthermore, cross-neutralization assays demonstrated that sera of convalescents from SARS-CoV-2 inhibited BANAL pseudovirus entry with an efficiency of approximately 80%, thereby highlighting conserved antigenic epitopes and informing the development of broad-spectrum therapeutic strategies against emerging SC2r-CoVs.
History
DepositionOct 22, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Spike glycoprotein
B: Spike glycoprotein
C: Spike glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)428,03060
Polymers415,2433
Non-polymers12,78757
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Spike glycoprotein / S glycoprotein / E2 / Peplomer protein


Mass: 138414.312 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Horseshoe bat sarbecovirus / Gene: S / Production host: Mammalia (mammals) / References: UniProt: A0AA49X976
#2: Sugar...
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 54 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Chemical ChemComp-EIC / LINOLEIC ACID / 9,12-LINOLEIC ACID


Mass: 280.445 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C18H32O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: SARS-CoV-2 related bat coronavirus BANAL-52 spike protein
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 450 kDa/nm / Experimental value: NO
Source (natural)Organism: Horseshoe bat sarbecovirus
Source (recombinant)Organism: Mammalia (mammals)
Details of virusType: VIRUS-LIKE PARTICLE
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.51 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20175 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.98 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002527216
ELECTRON MICROSCOPYf_angle_d0.462437017
ELECTRON MICROSCOPYf_chiral_restr0.0424356
ELECTRON MICROSCOPYf_plane_restr0.00324719
ELECTRON MICROSCOPYf_dihedral_angle_d4.48163912

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