Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Revealing the Structural Plasticity of SARS-CoV-2 nsp7 and nsp8 Using Structural Proteomics (HDX-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): Q Exactive HF

ORGANISM(S): Severe Acute Respiratory Syndrome Coronavirus 2

SUBMITTER: Valentine Courouble

LAB HEAD: Patrick Griffin

PROVIDER: PXD026066 | Pride | 2022-01-12

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Publications

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

Similar Datasets

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

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 | PXD033698 | Pride

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

2022-12-19 | PXD033702 | Pride

Patient-derived anti-Siglec-6 antibodies engineered into T cell-recruiting bispecific antibody formats for treating leukemia

Project description:Despite numerous therapeutic options, safe and curative therapy is out of reach for most chronic lymphocytic leukemia (CLL) patients. We previously reported the identification of a patient-derived CLL-binding antibody, JML-1, and identified Siglec-6 as the target of JML-1. Siglec-6 is an attractive target for cancer immunotherapy due to its absence on healthy tissues and potential role in immune signaling. In this work, we identify additional patient-derived anti-Siglec-6 antibodies, RC-1 and RC-2, that bind with higher affinity than JML-1, yet exhibit similar specificity and overlapping conformational epitopes. Both JML-1 and RC-1 were employed to generate anti-Siglec-6 x anti-CD3 T cell-recruiting bispecific antibodies (T-biAbs) which were effective in vitro at activating T cells and lysing target cells. Upon engineering RC-1 into more compact and rigid T-biAb formats, we achieved sub-pM EC50 values for cell lysis in vitro. In addition, the T-biAbs mediated the killing of primary CLL cells by autologous T cells at natural effector-to-target cell ratios ex vivo. The increased potency of this T-biAb format appears to be due to the reduction in the length and/or flexibility of the cytolytic synapse. Furthermore, the anti-Siglec-6 x anti-CD3 T-biAb was effective at curing mice in a systemic in vivo model of CLL.

2022-12-19 | PXD029601 | Pride

Selective modulation of nuclear receptor LRH-1 through target-ing buried and surface regions of the binding pocket

Project description:Liver receptor homolog-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviat-ing intestinal inflammation and disease states characterized by metabolic dysregulation in the liver. LRH-1 contains an unu-sually large ligand binding pocket, and generating synthetic modulators has been challenging. Leveraging a hexahydropen-talene (6HP) core, we have had recent success in generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding, while residues at the mouth of the pocket were targeted to mimic interactions made by endogenous phospholipids. Here, we unite these two designs into one hybrid mole-cule that is the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and struc-tural studies, we show that selective modulation can be driven through contacting deep vs. surface polar regions in the pock-et. While deep pocket contacts convey high-affinity, contacts with the pocket mouth dominate allostery and provide a phos-pholipid-like transcriptional response in cultured cells.

2022-05-04 | PXD031099 | Pride

Prion-like low complexity regions enable avid virus-host interactions during HIV-1 infection

Project description:Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short FG peptides to isolated capsid hexamers have been characterized, how these proteins engage biologically relevant extended HIV-1 capsid lattices is unknown. We have identified that prion-like low complexity regions (LCR) enable avid binding of CPSF6, NUP153 and SEC24C to curved hexameric capsid lattices. Structural studies reveal that LCR-LCR interactions mediate multivalent CPSF6 assembly, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. CPSF6 LCR mediated avid binding to HIV-1 cores is essential for functional virus-host interactions in infected cells. Investigational drug lenacapavir can access unoccupied hydrophobic pockets in the CPSF6-HIV-1 complex and potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results elucidate previously undescribed mechanisms of virus-host interactions and potent inhibition of HIV-1.

2022-08-31 | PXD030332 | Pride

(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains

Project description:Escherichia coli RelA is a ribosomal factor with strong (p)ppGpp synthesis activity that is dramatically activated in the presence of deacylated tRNA in the ribosomal A-site. RelA is a unique enzyme in that it is directly positively regulated by its product, alarmone nucleotide (p)ppGpp. Using HDX-MS, mulecular docking, biochemsitry, and microbiology approaches, we localise the (p)ppGpp binding site of E. coli RelA and uncover the molecular mechanism of RelA regulation by the alarmone.

2021-08-11 | PXD019953 | Pride

Structure dynamics of ApoA-I amyloidogenic variants in small HDL increase their ability to mediate cholesterol efflux

Project description:Specific mutations in Apolipoprotein A-I (ApoA-I) of high-density lipoprotein (HDL) are responsible for a late-onset systemic amyloidosis. Carriers do not exhibit increased cardiovascular disease risk despite reduced levels of ApoA-I/ HDL-cholesterol. To explain this paradox, we show that the HDL particle profile of L75P and L174S patients presents a higher relative abundance of the 8.4 nm vs 9.6 nm particles, and that serum from patients, as well as reconstituted 8.4 and 9.6 nm HDL particles (rHDL), possess increased capacity to catalyze cholesterol efflux from macrophages. Synchrotron radiation circular dichroism and hydrogen-deuterium exchange revealed that the variants in 8.4 nm rHDL have altered secondary structure composition and display a more flexible binding to lipids compared to their native counterpart. The reduced HDL-cholesterol levels of patients carrying ApoA-I amyloidogenic variants are thus balanced by higher proportion of small, dense HDL particles and better cholesterol efflux due to altered, region-specific protein structure dynamics.

2020-11-20 | PXD021266 | Pride

Pseudouridine-modified tRNA fragments repress malignant protein synthesis and predict leukemic progression in myelodysplastic syndrome

Project description:Transfer RNA (tRNA)-derived fragments (tRF) are emerging small noncoding (nc) RNAs that, while commonly altered in cancer, have poorly defined roles in tumorigenesis. Here we show that pseudouridylation (Ψ) of a stem-cell-enriched tRF subtype, mTOG, selectively inhibits malignant protein synthesis programs, thereby promoting engraftment and differentiation of myelodysplastic syndrome (MDS) hematopoietic stem and progenitor cells (HSPC). Building on evidence that mTOG-Ψ target the polyadenylate-binding protein cytoplasmic 1 (PABPC1), we employed HDX-MS to reveal critical interactions between mTOG and functional RNA-recognition motif (RRM) domains in PABPC1. Mechanistically, this hinders the recruitment of the translational co-activator PABPC1-interacting protein 1 (PAIP1) and strongly represses translation of transcripts sharing 5’UTR pyrimidine-enriched sequences (PES), including 5’ terminal oligopyrimidine tracts (TOP) that encode protein machinery components, and are frequently altered in cancer. Significantly, mTOG dysregulation leads to aberrantly increased 5’PES mRNA translation in malignant MDS-HSPC and is clinically associated with leukemic transformation and reduced patient survival. Taken together, these results define a critical role for tRF and Ψ in difficult-to-treat subsets of MDS characterized by high risk of progression to acute myeloid leukemia.

2021-12-20 | PXD023122 | Pride

SMARCA2, VHL Binary and Ternary Complex formation with ACBi1, PROTAC1, and PROTAC2

Project description:Interrogration of impacts on Hydrogen Deuterium Exchange of ligand binding using heterobifunctional molecules ACBi1(SiTX-0038406), PROTAC1(SiTX-0038404) and PROTAC2(SiTX-0038405) on target proteins SMARCA2 and VHL both in the binary and Ternary modes.

2022-10-12 | PXD033849 | Pride

An HDX-MS optimised approach to characterise nanobodies as tools for biochemical and structural studies

Project description:Given the far-reaching applications of nanobodies, a stream-lined approach to characterizing nanobodies tailored to its intended application is warranted. Camelids can produce a large number of antibodies that can bind to a host of epitopes spanning the entire protein of interest, making identification of binders appropriate to your study slow and cumbersome. To overcome this challenge, we propose the use of hydrogen-deuterium mass spectrometry (HDX-MS) to rapidly characterize nanobody epitopes. HDX-MS is a technique that measures the rate of exchange of amide hydrogens in the protein backbone and can provide valuable insight into interaction surfaces with binding partners. Using the example of the lipid signalling complex PI3K, we employed HDX-MS to characterize the epitopes of a large cohort of nanobodies simultaneously with reasonable accuracy. Some of these nanobodies proved to be extremely useful, stabilizing a flexible region in structural studies and blocking signalling inputs from activating partners in in-vitro kinase assays. These experiments were in turn, instrumental in obtaining the first high resolution structure of a PI3K complex, besides providing novel tools to study PI3K signalling in-vivo.

2021-09-22 | PXD025207 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data