1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms of the macrodomain of SARS-CoV-2 non-structural protein 3b

Francesca Cantini ,Christin Fuks ,Aikaterini C Tsika ,Nikolaos K Fourkiotis ,Frank Löhr ,Felicitas Kutz ,Jasleen Kaur Bains ,Harald Schwalbe ,Sophie Marianne Korn ,Nusrat Shahin Qureshi ,Martin Hengesbach ,Jens Wöhnert ,Anna Wacker ,Christian Richter ,Nina Kubatova ,V.l Linhard ,Lucia Banci ,Karthikeyan Dhamotharan ,S.l Gande ,Julia E Weigand ,Nadide Altincekic ,Sridhar Sreeramulu ,Marie Hutchison ,Krishna Saxena ,Dennis J Pyper ,Bruno Hargittay ,Jan‐Niklas Tants ,Andreas Schlundt ,Nathalie Meiser ,Boris Fürtig ,Georgios A Spyroulias

doi:10.1007/s12104-020-09973-4

Abstract

The SARS-CoV-2 genome encodes for approximately 30 proteins. Within the international project COVID19-NMR, we distribute the spectroscopic analysis of the viral proteins and RNA. Here, we report NMR chemical shift assignments for the protein Nsp3b, a domain of Nsp3. The 217-kDa large Nsp3 protein contains multiple structurally independent, yet functionally related domains including the viral papain-like protease and Nsp3b, a macrodomain (MD). In general, the MDs of SARS-CoV and MERS-CoV were suggested to play a key role in viral replication by modulating the immune response of the host. The MDs are structurally conserved. They most likely remove ADP-ribose, a common posttranslational modification, from protein side chains. This de-ADP ribosylating function has potentially evolved to protect the virus from the anti-viral ADP-ribosylation catalyzed by poly-ADP-ribose polymerases (PARPs), which in turn are triggered by pathogen-associated sensing of the host immune system. This renders the SARS-CoV-2 Nsp3b a highly relevant drug target in the viral replication process. We here report the near-complete NMR backbone resonance assignment (1H, 13C, 15N) of the putative Nsp3b MD in its apo form and in complex with ADP-ribose. Furthermore, we derive the secondary structure of Nsp3b in solution. In addition, 15N-relaxation data suggest an ordered, rigid core of the MD structure. These data will provide a basis for NMR investigations targeted at obtaining small-molecule inhibitors interfering with the catalytic activity of Nsp3b.

Highlights

Severe acute respiratory syndrome coronavirus 2 (SARSCoV-2), the cause of the pandemic that began in early 2020 and is accompanied by the respiratory disease COVID-19, is the latest member of a Coronaviridae clade, which includes SARS-CoV from 2002 and the Middle east respiratory syndrome (MERS)-CoV
Besides the membrane-bound Spike protein, which is important for the entry of the virus into the cell, a number of non-structural proteins (Nsps) such as the two proteases Nsp5 (Mpro) and Nsp3d (PLpro), the Nsp3b ADP-ribose-phosphatase macrodomain (MD), and the Nsp7/8/12 RNA-dependent RNA polymerase complex are obvious drug targets
Starting from the N-terminus, its individual functional domains are named Nsp3a to Nsp3e followed by the ectodomain, which is embedded between two transmembrane regions, and the C-terminal CoV-Y domain

Summary

Nsp3b-ADP-ribose 1H-15N-HSQC

1H, 13C, and 15N backbone chemical shift assignments of the apo and the ADP-ribose bound forms. The research consortium COVID19-NMR, which was founded at the end of March 2020, rapidly and publicly supports the search for antiviral drugs by enabling an NMRbased screening approach. This requires the large-scale production of all drugable proteins and RNAs of SARS-CoV-2, as well as an extensive assignment of their NMR resonances and the determination of their structures as a prerequisite for rational drug design. We provide here the near-complete backbone assignment of the SARS-CoV-2 Nsp3b MD and thereby enable its exploitation in subsequent applications, such as drug screening and interaction mapping with amino acid resolution

Methods and experiments

Findings

Compliance with ethical standards