Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

Zhang, Ying; Wecksler, Aaron T.; Molina, Patricia G.; Deperalta, Galahad; Gross, Michael L.

doi:10.1007/s13361-017-1601-7

Cited by 53 publications

(60 citation statements)

References 14 publications

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“…5). The degree of protection is consistent with prior observations of antibody-antigen interactions by hydroxyl radical footprinting (19). Both M177 and H207 are greater than 40 Å distant from the ACE2 binding site on the RBD, suggesting that Nb3 may inhibit Spike-ACE2…”

Section: Nb3 Interacts With the Spike S1 Domain External To The Rbdsupporting

confidence: 88%

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

Schoof

Faust

Saunders

et al. 2020

Preprint

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Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

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Section: Nb3 Interacts With the Spike S1 Domain External To The Rbdsupporting

confidence: 88%

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

Schoof

Faust

Saunders

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Two neighboring surface residues on the S1 N-terminal domain of Spike (M177 and H207) were protected in the presence of Nb3 at a level consistent with prior observations of antibody-antigen interactions by hydroxyl radical footprinting ( fig. S5) (19). Previously discovered coronavirus neutralizing antibodies bind an epitope within the N-terminal domain of Spike with Fab fragments that are non-competitive with the host cell receptor (20,21).…”

mentioning

confidence: 99%

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

Schoof

Faust

Saunders

et al. 2020

Science

386

443

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The SARS-CoV-2 virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryogenic electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

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“…Alternatively, a “top-down” approach using high-resolution MS coupled with powerful fragmentation technologies is becoming increasingly significant for the comprehensive structural analysis of intact proteins without proteolytic digestion 3–5 . The advantages of top-down MS to characterize the structures of large proteins and to interrogate post-translational modifications and proteoforms has been demonstrated 6–8 . Coupling native MS with top-down MS allows the higher order structures of protein complexes to be probed 9,10 .…”

mentioning

confidence: 99%

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels

Takemori

Wongkongkathep

et al. 2017

Anal. Chem.

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Biologists’ preeminent toolbox for separating, analyzing, and visualizing proteins is SDS-PAGE, yet recovering the proteins embedded in these polyacrylamide media as intact species is a long-standing challenge for mass spectrometry. In conventional workflows, protein mixtures from crude biological samples are electrophoretically separated at high-resolution within N,N′-methylene-bis-acrylamide crosslinked polyacrylamide gels to reduce sample complexity and facilitate sensitive characterization. However, low protein recoveries, especially for high molecular weight proteins, often hinder characterization by mass spectrometry. We describe a workflow for top-down/bottom-up mass spectrometric analyses of proteins in polyacrylamide slab gels using dissolvable, bis-acryloylcystamine-crosslinked polyacrylamide, enabling high-resolution protein separations while recovering intact proteins over a broad size range efficiently. The inferior electrophoretic resolution long associated with reducible gels has been overcome, as demonstrated by SDS-PAGE of crude tissue extracts. This workflow elutes intact proteins efficiently, supporting MS and MS/MS from proteins resolved on biologists’ preferred separation platform.

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Mapping the Binding Interface of VEGF and a Monoclonal Antibody Fab-1 Fragment with Fast Photochemical Oxidation of Proteins (FPOP) and Mass Spectrometry

Cited by 53 publications

References 14 publications

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike

Top-down/Bottom-up Mass Spectrometry Workflow Using Dissolvable Polyacrylamide Gels

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