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

J Am Soc Mass Spectrom. 2021 Jul 7;32(7):1618-1630. doi: 10.1021/jasms.1c00086. Epub 2021 Jun 14.

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 nsp7:nsp8 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 nsp7:nsp8 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 nsp7:nsp8 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 nsp7:nsp8 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 nsp7:nsp8 crystal structure. Instead, our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without significantly altering nsp7-nsp8 interactions.

MeSH terms

  • COVID-19 / virology
  • Coronavirus RNA-Dependent RNA Polymerase / chemistry*
  • Coronavirus RNA-Dependent RNA Polymerase / genetics
  • Coronavirus RNA-Dependent RNA Polymerase / metabolism
  • Humans
  • Hydrogen Deuterium Exchange-Mass Spectrometry
  • Models, Molecular
  • Protein Conformation
  • Proteomics / methods*
  • SARS-CoV-2 / chemistry
  • Viral Nonstructural Proteins / chemistry*
  • Viral Nonstructural Proteins / genetics
  • Viral Nonstructural Proteins / metabolism

Substances

  • NS8 protein, SARS-CoV-2
  • Viral Nonstructural Proteins
  • Coronavirus RNA-Dependent RNA Polymerase
  • NSP7 protein, SARS-CoV-2