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.
Subject(s)
Coronavirus RNA-Dependent RNA Polymerase/chemistry , Proteomics/methods , Viral Nonstructural Proteins/chemistry , COVID-19/virology , Coronavirus RNA-Dependent RNA Polymerase/genetics , Coronavirus RNA-Dependent RNA Polymerase/metabolism , Humans , Hydrogen Deuterium Exchange-Mass Spectrometry , Models, Molecular , Protein Conformation , SARS-CoV-2/chemistry , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolismABSTRACT
The novel severe respiratory syndrome-like coronavirus (SARS-CoV-2) causes COVID-19 in humans and is responsible for one of the most destructive pandemics of the last century. At the root of SARS-CoV infection is the interaction between the viral spike protein and the human angiotensin converting enzyme 2 protein, which allows the virus to gain entry into host cells through endocytosis. In this work, we apply hydrogen-deuterium exchange mass spectrometry (HDX-MS) to provide a detailed view of the functional footprint and conformational dynamics associated with this interaction. Our results broadly agree with the binding interface derived from high resolution X-ray crystal structure data but also provide insights into shifts in structure and dynamics that accompany complexation, including some that occur immediately outside of the core binding interface. We propose that dampening of these "binding-site adjacent" dynamic shifts could represent a mechanism for neutralizing activity in a multitude of spike protein-targeted mAbs that have been found to specifically bind these "peripheral" sites. Our results highlight the unique capacity of HDX-MS to detect potential neutralization "hotspots" outside of the core binding interfaces defined by high resolution structural data.