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Revealing the Structural Plasticity of SARS-CoV-2 nsp7 and nsp8 Using Structural Proteomics.
Courouble, Valentine V; Dey, Sanjay Kumar; Yadav, Ruchi; Timm, Jennifer; Harrison, Jerry Joe E K; Ruiz, Francesc X; Arnold, Eddy; Griffin, Patrick R.
  • Courouble VV; Department of Molecular Medicine, The Scripps Research Institute, Jupiter, Florida 33458, United States.
  • Dey SK; Center for Advanced Biotechnology & Medicine, and Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States.
  • Yadav R; Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India.
  • Timm J; Center for Advanced Biotechnology & Medicine, and Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States.
  • Harrison JJEK; Center for Advanced Biotechnology & Medicine, and Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States.
  • Ruiz FX; Center for Advanced Biotechnology & Medicine, and Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States.
  • Arnold E; Department of Chemistry, School of Physical and Mathematical Sciences, University of Ghana, Box LG 56, Legon, Accra, Ghana.
  • Griffin PR; Center for Advanced Biotechnology & Medicine, and Department of Chemistry & Chemical Biology, Rutgers University, Piscataway, New Jersey 08854, United States.
J Am Soc Mass Spectrom ; 32(7): 1618-1630, 2021 Jul 07.
Article in English | MEDLINE | ID: covidwho-1267989
ABSTRACT
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 nsp7nsp8 complex has been published. The current understanding of this complex is based on structures from truncated constructs, with missing electron densities, or from related CoV species where SARS-CoV-2 nsp7 and nsp8 share upward of 90% sequence identity. Despite available structures solved using crystallography and cryo-EM representing detailed static snapshots of the nsp7nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the individual proteins and how they complex to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and cross-linking mass spectrometry (XL-MS), illuminate the dynamics of SARS-CoV-2 full-length nsp7 and nsp8 proteins and the nsp7nsp8 protein complex. Results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of the SARS-CoV-2 truncated nsp7nsp8 complex. Furthermore, mapping of XL-MS data onto higher-order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7nsp8 crystal structure. Instead, our results suggest that the nsp7nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without significantly altering nsp7-nsp8 interactions.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Viral Nonstructural Proteins / Proteomics / Coronavirus RNA-Dependent RNA Polymerase Type of study: Prognostic study / Randomized controlled trials Limits: Humans Language: English Journal: J Am Soc Mass Spectrom Year: 2021 Document Type: Article Affiliation country: Jasms.1c00086

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Viral Nonstructural Proteins / Proteomics / Coronavirus RNA-Dependent RNA Polymerase Type of study: Prognostic study / Randomized controlled trials Limits: Humans Language: English Journal: J Am Soc Mass Spectrom Year: 2021 Document Type: Article Affiliation country: Jasms.1c00086