Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

Silva, Rui P.; Huang, Yimin; Nguyen, Annalee W.; Hsieh, Ching-Lin; Olaluwoye, Oladimeji S; Kaoud, Tamer S.; Wilen, Rebecca E; Qerqez, Ahlam; Park, Jun-Gyu; Khalil, Ahmed Magdy; Azouz, Laura R.; Le, Kevin; Bohanon, Amanda L; DiVenere, Andrea M.; Liu, Yutong; Lee, Alison G.; Amengor, Dzifa; Shoemaker, Sophie R.; Costello, Shawn M.; Padlan, Eduardo A.; Marqusee, Susan; Martínez-Sobrido, Luis; Dalby, Kevin N.; D’Arcy, Sheena; McLellan, Jason S.; Maynard, Jennifer A.

doi:10.7554/elife.83710

Cited by 35 publications

(39 citation statements)

References 95 publications

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“…2e) . Consistently, previous HDX-MS studies using the recombinant ectodomain of the spike protein with a prefusion stabilization (2P mutations) suggested the minor presence of an alternative conformation, in which S2 residues are solvent exposed, which can be targeted by nAbs, such as 3A3 and RAY53 22,23,25 . Therefore, in active viruses, the typical closed/packed prefusion conformation is dominant but the spike protein itself has ability to spontaneously transit to an open-trimer conformation.…”

Section: Mainsupporting

confidence: 79%

Unprecedented spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

Fukuhara,

Dokainish,

Kita

et al. 2023

Preprint

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Understanding the molecular properties of SARS-CoV-2 is crucial to tackle the continuing variant emergence as well as future outbreaks. Current structural knowledge of the trimeric spike protein is largely based on truncated recombinant proteins and inactivated full-length forms, which may suffer from overstabilization through designed mutations and chemical fixation. Here, we apply cryo-electron tomography (cryo-ET) at a Biosafety level 3 facility to study the virus structure in its native, active state. The virus particles show variable shapes with diffusible spikes. While the majority of spike proteins display typical prefusion trimer conformations, we identify atypical open-trimer prefusion states, revealing a hidden level of flexibility. The sub-tomogram averaged structure also suggests a loosely packed trimer structure. Spike dynamics reveal cryptic epitopes in conserved regions among coronaviruses that can be targeted for broadly effective vaccines. Therefore, the structural analysis of virus structures in their active forms will provide significant implications on fusion mechanism as well as vaccine and antibody/drug design.

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Section: Mainsupporting

confidence: 79%

Unprecedented spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

Fukuhara,

Dokainish,

Kita

et al. 2023

Preprint

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“…This conserved pocket is shared among Omicron variants and is located at the critical functional hinge region involved in modulation S1-S2 opening motions. The experimental studies indicated that S dynamics may impact hinge epitope accessibility [46] and our results showed that this cryptic epitope may be accessible for ligand binding in the S trimer complexes with ACE2.…”

Section: Resultsmentioning

confidence: 62%

“…In this context, it may be worth noting the results of HDX-MS experiments suggesting partial destabilization not only near the S1/S2 cleavage site but also in the HR1 region which may be functionally required to promote fusion stage. Our results showed considerable stability of the highly conserved hinge epitope (residues 980-1010) located in the S2 region between the CH and HR1 segments that is involved in regulation of conformational changes required for fusion of the viral envelope [46].…”

Section: Resultsmentioning

confidence: 99%

“…This pocket is formed by conserved residues from structurally stable UH region (residues 736-781) and CH region (residues 986-1035). Strikingly, the predicted inter-protomer S2 pocket overlaps with the highly conserved, conformational hinge epitope spanning residues 970–1006 of the SARS-2 spike, at the apex of the S2 domain [46]. This conserved pocket is shared among Omicron variants and is located at the critical functional hinge region involved in modulation S1-S2 opening motions.…”

Section: Resultsmentioning

confidence: 99%

“…The recent structural investigations unveiled another cryptic binding pocket located in the NTD region, also demonstrating that tetrapyrrole products of heme metabolism, biliverdin and bilirubin, can bind to this site with nanomolar affinity and inhibit neutralizing activity of the NTD antibodies recognizing these epitopes [43–45]. By leveraging HDX-MS and antibody engineering approaches, structural studies confirmed a cryptic hinge epitope located in the S2 region between the CH and HR1 segments that play a critical role in the spike conformational changes required for fusion of the viral envelope and target cell membrane [46]. This S2 hinge epitope is partially occluded by the S1 domain and access to this epitope may be controlled through RBD conformational switching.…”

Section: Introductionmentioning

confidence: 99%

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Examining Functional Linkages Between Conformational Dynamics, Protein Stability and Evolution of Cryptic Binding Pockets in the SARS-CoV-2 Omicron Spike Complexes with the ACE2 Host Receptor: Recombinant Omicron Variants Mediate Variability of Conserved Allosteric Sites and Binding Epitopes

Alshahrani,

Gupta,

Xiao

et al. 2023

Preprint

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In the current study, we explore coarse-grained simulations and atomistic molecular dynamics together with binding energetics scanning and cryptic pocket detection in a comparative examination of conformational landscapes and systematic characterization of allosteric binding sites in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB.1 spike full-length trimer complexes with the host receptor ACE2. Microsecond simulations, Markov state models and mutational scanning of binding energies of the SARS-CoV-2 BA.2 and BA.2.75 receptor binding domain complexes revealed the increased thermodynamic stabilization of the BA.2.75 variant and significant dynamic differences between these Omicron variants. Molecular simulations of the SARS-CoV-2 Omicron spike full length trimer complexes with the ACE2 receptor complemented atomistic studies and enabled an in-depth analysis of mutational and binding effects on conformational dynamic and functional adaptability of the Omicron variants. Despite considerable structural similarities, Omicron variants BA.2, BA.2.75 and XBB.1 can induce unique conformational dynamic signatures and specific distributions of the conformational states. Using conformational ensembles of the SARS-CoV-2 Omicron spike trimer complexes with ACE2, we conducted a comprehensive cryptic pocket screening to examine the role of Omicron mutations and ACE2 binding on the distribution and functional mechanisms of the emerging allosteric binding sites. This analysis captured all experimentally known allosteric sites and discovered networks of inter-connected and functionally relevant allosteric sites that are governed by variant-sensitive conformational adaptability of the SARS-CoV-2 spike structures. The results detailed how ACE2 binding and Omicron mutations in the BA.2, BA.2.75 and XBB.1 spike complexes modulate the distribution of conserved and druggable allosteric pockets harboring functionally important regions. The results of are significant for understanding functional roles of druggable cryptic pockets that can be used for allostery-mediated therapeutic intervention targeting conformational states of the Omicron variants.

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Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

Haynes,

Keppel,

Mekonnen

et al. 2024

Rapid Comm Mass Spectrometry

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RationaleHydrogen/deuterium exchange mass spectrometry (HDX‐MS) can provide precise analysis of a protein's conformational dynamics across varied states, such as heat‐denatured versus native protein structures, localizing regions that are specifically affected by such conditional changes. Maximizing protein sequence coverage provides high confidence that regions of interest were located by HDX‐MS, but one challenge for complete sequence coverage is N‐glycosylation sites. The deuteration of peptides post‐translationally modified by asparagine‐bound glycans (glycopeptides) has not always been identified in previous reports of HDX‐MS analyses, causing significant sequence coverage gaps in heavily glycosylated proteins and uncertainty in structural dynamics in many regions throughout a glycoprotein.MethodsWe detected deuterated glycopeptides with a Tribrid Orbitrap Eclipse mass spectrometer performing data‐dependent acquisition. An MS scan was used to identify precursor ions; if high‐energy collision‐induced dissociation MS/MS of the precursor indicated oxonium ions diagnostic for complex glycans, then electron transfer low‐energy collision‐induced dissociation MS/MS scans of the precursor identified the modified asparagine residue and the glycan's mass. As in traditional HDX‐MS, the identified glycopeptides were then analyzed at the MS level in samples labeled with D2O.ResultsWe report HDX‐MS analysis of the SARS‐CoV‐2 spike protein ectodomain in its trimeric prefusion form, which has 22 predicted N‐glycosylation sites per monomer, with and without heat treatment. We identified glycopeptides and calculated their average isotopic mass shifts from deuteration. Inclusion of the deuterated glycopeptides increased sequence coverage of spike ectodomain from 76% to 84%, demonstrated that glycopeptides had been deuterated, and improved confidence in results localizing structural rearrangements.ConclusionInclusion of deuterated glycopeptides improves the analysis of the conformational dynamics of glycoproteins such as viral surface antigens and cellular receptors.

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Identification of a conserved S2 epitope present on spike proteins from all highly pathogenic coronaviruses

Cited by 35 publications

References 95 publications

Unprecedented spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

Unprecedented spike flexibility revealed by BSL3 Cryo-ET of active SARS-CoV-2 virions

Examining Functional Linkages Between Conformational Dynamics, Protein Stability and Evolution of Cryptic Binding Pockets in the SARS-CoV-2 Omicron Spike Complexes with the ACE2 Host Receptor: Recombinant Omicron Variants Mediate Variability of Conserved Allosteric Sites and Binding Epitopes

Inclusion of deuterated glycopeptides provides increased sequence coverage in hydrogen/deuterium exchange mass spectrometry analysis of SARS‐CoV‐2 spike glycoprotein

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