Structural insights into the activity regulation of full-length non-structural protein 1 from SARS-CoV-2 and substrate recruitment by the hexameric MecA-ClpC complex

authored by
Ying Wang
supervised by
Teresa Carlomagno

Since the outbreak of the COVID-19 pandemic in December 2019, SARS-CoV-2 has generated awareness for the requirement of novel antiviral drugs that target new proteins. Previous studies have pointed to the pathogenic significance of the non-structural protein 1 (Nsp1), which was proposed to be a major virulence factor due to its dual role in host translation inhibition and viral replication of SARSCoV- 2. The precise mechanisms of these two functions of Nsp1 are still uncovered. Here, I report the backbone chemical-shift assignments and the atomic-resolution NMR structure of full-length Nsp1 from SARS-CoV-2 solved by NMR. I found that Cov-2 Nsp1 consists of a folded N-terminal domain and an unfolded C-terminal region. Previous studies have identified a surface of the folded N-terminal domain of Nsp1 that associates with both host mRNA, in its function as inhibitor of the host protein translation, and the viral 5’-UTR RNA, in its function as promoter of viral protein translation. I found that the acidic C-terminal tail of Nsp1 folds back on this surface and masks the RNA binding site. A recent Cryo-EM study has shown that the end of C-terminal region of Nsp1 interacts with the mRNA entry tunnel on the 40S subunit of the ribosome, thus inhibiting host mRNA entry and promoting its degradation. I propose that the RNA binding site on the Nsp1 N-terminal domain is protected by its C-terminal tail before Nsp1 contacts the ribosome. Upon ribosome binding, the C-terminal tail is displaced, and the RNA binding site is exposed to recruit either host mRNA or the viral 5’ UTR. My findings have consequences for the design of drugs targeting the RNA binding surface of Nsp1, as it demonstrates the need to develop a molecule that can not only recognize this surface with high affinity, but also displace the C-terminal tail to render this surface accessible. In bacteria, the unfoldase ClpC is a member of the conserved Hsp100/Clp family of AAA+ ATPases and is involved in various cellular processes. The functional form of ClpC is an hexameric assembly, which is responsible for controlled unfolding of substrate proteins. The ClpC hexamer can further associate with the protease ClpP to form a complete protein degradation machine. MecA functions as an adaptor protein of ClpC and is necessary to both promote the formation of the functional ClpC hexamer and to recruit specific substrate proteins, such as the transcription factor ComK. Degradation of ComK via MecA-mediated recruitment to the ClpCP complex is part of the regulatory mechanisms of the development of cell competence. The mechanisms of selective substrate recruitment are still unknown, due to the conspicuous conformational dynamics and heterogeneity that characterize this step. To understand how the substrate ComK is recruited to the MecA–ClpC complex, I reconstituted the ComK– MecA–ClpC complex in vitro and applied nuclear magnetic resonance (NMR) spectroscopy and other biophysical techniques, such as multi-angle light scattering. I found that addition of ComK stabilizes the MecA–ClpC complex by forming a homogeneous ternary protein complex, which contains a ClpC hexamer, four MecA and two ComK molecules in the presence of ATP. The structural differences between the MecA–ClpC and the ComK–MecA–ClpC complexes were also monitored by small-angle X-ray scattering datasets, which furthered confirmed the presence of the interaction between ComK and MecA.

Section Structural Biology
Doctoral thesis
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