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Revealing the Structural Plasticity of SARS-CoV-2 nsp7 and nsp8 Using Structural Proteomics (XL-MS)

ABSTRACT: SARS-CoV-2 nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase and other nspsHere we used solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the dynamics of SARS-CoV-2 full-length nsp7, nsp8, and nsp7:nsp8 proteins and protein complex.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Severe Acute Respiratory Syndrome Coronavirus 2

SUBMITTER: Valentine Courouble

LAB HEAD: Patrick R. Griffin

PROVIDER: PXD026191 | Pride | 2022-01-12

REPOSITORIES: Pride

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			Action	DRS
	202001216_nsp7_trypsin_NoXL_REP1.raw	Raw
	202001216_nsp7_trypsin_NoXL_REP2.raw	Raw
	202001216_nsp7_trypsin_XL_REP1.raw	Raw
	202001216_nsp7_trypsin_XL_REP2.raw	Raw
	202001216_nsp7_trypsin_XL_REP3.raw	Raw

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Revealing the Structural Plasticity of SARS-CoV-2 nsp7 and nsp8 Using Structural Proteomics.

Courouble Valentine V VV Dey Sanjay Kumar SK Yadav Ruchi R Timm Jennifer J Harrison Jerry Joe E K JJEK Ruiz Francesc X FX Arnold Eddy E Griffin Patrick R PR

Journal of the American Society for Mass Spectrometry 20210614 7

Coronavirus (CoV) nonstructural proteins (nsps) assemble to form the replication-transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase and other nsps. To date, no structure of the full-length SARS-CoV-2 nsp7:nsp8 complex has been published. The current understanding of this complex is based on structures from truncated constructs, with missing electron densities ...[more]

PMID: 34121407

Publication: 1/2

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Biochemical and structural insights into SARS-CoV-2 polyprotein processing by Mpro (XL-MS)

Project description:SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic, entailing enormous disruption worldwide. Its ~30 kb RNA genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main protease (Mpro/nsp5). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-10/11 polyprotein, whose mature products are cofactors of the viral replicase, identifying the order of cleavages: 1) nsp9-10, 2) nsp8-9/nsp10-11, and 3) nsp7-8. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and X-ray scattering yielded three-dimensional models of the nsp7-10/11 polyprotein, and the data suggest that the nsp7-10/11 structure in complex with Mpro strongly resembles the unbound polyprotein. Our array of techniques supports the notion that interplay between polyprotein conformation and junction accessibility determine the preference and order of cleavages. Finally, we leveraged assays of limited proteolysis by Mpro of nsp7-11 using a series of inhibitors/binders to characterize Mpro inhibition using a polyprotein substrate.

2022-12-19 | PXD033748 | Pride

Selennoprotein S interacts with the replication and transcription complex of the SARS-CoV-2 by binding nsp7

Project description:The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replicates and evades detection by using endoplasmic reticulum (ER) membranes and their associated protein machineries. Among these hijacked human ER proteins is selenoprotein S, which usually takes part in the ER protein degradation pathway, NFkB signaling, and regulation of cytokines secretion. While the role of selenoprotein S in the viral life cycle is not yet known, it has been reported that it interacts with SARS-CoV-2 non-structural protein 7 (nsp7), a viral protein essential for the reproduction of SARS-CoV-2. However, it was unknown if selenoprotein S and nsp7 interact directly and whether the two proteins can still interact when nsp7 is bound to the virus' replication machinery. Using biochemical assays, we show that selenoprotein S indeed binds directly to nsp7. In addition, we find that selenoprotein S can also bind nsp7 when it is in a complex with the coronavirus's minimal replication complex: nsp7, nsp8 and the RNA-dependent RNA polymerase. Using cross-linking experiments, we mapped the interactions of selenoprotein S and nsp7 in the replication complex and show that the hydrophobic segment of selenoprotein S is essential for binding nsp7. This arrangement leaves an extended helix and the intrinsically disordered segment of selenoprotein S exposed and free to potentially recruit additional proteins to the replication complex. Thus, selenoprotein S is shown to directly interact with the viral replication complex, which places this human protein at the heart of viral replication.

2023-03-11 | PXD038629 | Pride

Structural basis for heme-dependent NCoR binding to the transcriptional repressor REV-ERBβ

Project description:Heme is the endogenous ligand for the constitutively repressive REV-ERB nuclear receptors, REV-ERBα (NR1D1) and REV-ERBβ (NR1D2), but how heme regulates REV-ERB activity remains unclear. While cellular studies indicate heme is required for the REV-ERBs to bind the corepressor NCoR and repress transcription, fluorescence-based biochemical assays and crystal structures suggest that heme displaces NCoR. Here, we found that heme artifactually influences detection of NCoR interaction in fluorescence-based assays. Using fluorescence-independent methods, including isothermal titration calorimetry, NMR spectroscopy, and XL-MS, we determined that heme remodels the thermodynamic profile of NCoR binding to REV-ERBβ ligand-binding domain (LBD) and directly increases LBD binding affinity for an NCoR interaction motif. We further report two crystal structures of REV-ERBβ LBD cobound to heme and NCoR peptides, which reveal the heme-dependent NCoR binding mode. By resolving previous contradictory biochemical, structural, and cellular studies, our findings should facilitate renewed progress toward understanding heme-dependent REV-ERB activity.

2021-02-24 | PXD021761 | Pride

XL-MS Analysis of Recombinant GPR158:RGS7:Gb5 Complex in Detergent Micelles

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2023-03-11 | PXD026603 | Pride

Optimized parameter settings enhance proteome-wide formation of crosslinks on low-abundant proteins

Project description:Studies using crosslinking coupled to mass spectrometry on the proteome-wide level have spurred great interest as they facilitate structural probing of protein interactions in living cells or even organisms. Here we show, by using both an in-vitro mimic of a crowded cellular environment and eukaryotic cell lysates, that current proteome-wide crosslinking protocols have a bias for high abundant proteins. We demonstrate that this bias can be explained by kinetics that govern the formation of a crosslink between two polypeptides. We further show that optimized parameter settings, in particularly an excess of crosslinker, leadto a significant overall increase in the detection of lower abundant proteins within cellular lysates on a proteome-wide scale. Our study therefore explains the cause of a major limitation in current proteome-wide crosslinking studies and demonstrates a way forward how to address a larger part of the proteome by crosslinking

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Dynamics of different mGBP as revealed by computational and experimental methods

Project description:Guanylate binding proteins (GBP) belong to the superfamily of dynamin proteins. Those might assemble into larger structures upon initial dimerization. We analyzed monomeric and dimerization of mGBP7 via molecular dynamic simulations and compared the developed models with data from crosslinking mass spectrometry experiments.

2024-01-26 | PXD026979 | Pride

Revealing the Structural Plasticity of SARS-CoV-2 nsp7 and nsp8 Using Structural Proteomics (HDX-MS)

Project description:SARS-CoV-2 nsp7 and nsp8 are important cofactors of the RTC, as they interact and regulate the activity of RNA-dependent RNA polymerase and other nspsHere we used solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the dynamics of SARS-CoV-2 full-length nsp7, nsp8, and nsp7:nsp8 proteins and protein complex.

2022-01-12 | PXD026066 | Pride

Structure of Anabaena flos-aquae gas vesicles revealed by cryo-ET

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Histone interaction landscapes visualized by crosslinking mass spectrometry in intact cell nuclei

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