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bioRxiv · 10.1101/2021.03.06.434214

Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics

Courouble, V.·Dey, S.·Yadav, R.·Timm, J.·Harrison, J.·Ruiz, F. X.·Arnold, E.·Griffin, P. R.
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Abstract

Coronavirus (CoV) non-structural 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 (RdRp) and other nsps. To date, no structure of full-length SARS-CoV-2 nsp7:nsp8 complex has been published. Current understanding of this complex is based on structures from truncated constructs or with missing electron densities and complexes from related CoV species with which SARS-CoV-2 nsp7 and nsp8 share upwards of 90% sequence identity. Despite available structures being solved using crystallography and cryo-EM representing detailed 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 complex and how it assembles to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen-deuterium exchange mass spectrometry (HDX-MS) and crosslinking mass spectrometry (XL-MS), illuminate the structural dynamics of the SARS-CoV-2 full-length nsp7:nsp8 complex. The results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of 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 altering nsp7-nsp8 interactions.

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Courouble, V., Dey, S., Yadav, R., Timm, J., Harrison, J., Ruiz, F. X., Arnold, E., Griffin, P. R.. 2021-03-06. Resolving the Dynamic Motions of SARS-CoV-2 nsp7 and nsp8 Proteins Using Structural Proteomics. https://doi.org/10.1101/2021.03.06.434214

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