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.
