Structure, mechanism and crystallographic fragment screening of the SARS-CoV-2 NSP13 helicase

Joseph A Newman,Frank Von Delft,Antony Aimon,José Brandão-Neto,Yuliana Yosaatmadja,Setayesh Yadzani,Rachael Skyner,Daren Fearon,Matthieu Schapira,Alice Douangamath,Opher Gileadi,Louise Dunnett,Tyler Gorrie-Stone

doi:10.1038/s41467-021-25166-6

Abstract

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two “druggable” pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.

Highlights

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic
We identified two pockets of interest that are expected to be functionally relevant (Fig. 6a): the binding site occupied by AMPPNP, at the interface of domains 1A and 2A, and a pocket lined by domains 1A, 1B, and 2A which is occupied by the 5′-end of the substrate RNA in the SARS-Cov-2 transcription complex [PDB:7CXM]19
In a systematic analysis of the sequence conservation of nineteen binding pockets from fifteen SARS-CoV-2 proteins across the same twenty-seven coronaviruses, we find that the active site of RdRp/NSP12 (94% sidechains conserved) and the ADP-bound pocket of the NSP12 NiRAN domain (87% sidechains conserved) are the only two cavities with a degree of conservation as high as the 5′RNA site of Non-structural protein 13 (NSP13) (Yazdani et al bioRxiv 2021)

Summary

Introduction

There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. We present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. NSP13 contains 5 domains, a N-terminal Zinc binding domain (ZBD) that coordinates 3 structural Zinc ions, a helical “stalk” domain, a beta-barrel 1B domain and two “RecA like” helicase subdomains 1 A and 2 A that contain the residues responsible for nucleotide binding and hydrolysis This same basic 5-domain architecture is shared by by other Nidovirus helicases such as the NSP10 proteins from Equine arteritis virus[15] and Porcine reproductive and respiratory syndrome virus[16] and to a lesser extent the human nonsense-mediated mRNA decay factor UPF117, which feature a structurally similar helicase core

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Results

Conclusion