ABSTRACT
SARS-CoV-2 main protease (SARS-CoV-2 Mpro) is a cysteine protease that hydrolyses the viral polyproteins at several sites with a preference for the Leu-Gln(Ser, Ala, Gly) sequences1. The enzyme represents one of the main drug-target candidates for covid-19 syndrome because the large and deep pocket at the active site and its crucial activity for viral replication2-5 Here, we provide X-ray structural data on SARS-CoV-2 Mpro in complex with the isolated Zn2+ ion. The comparison with the apo SARSCoV- 2 Mpro shows that residues involved in zinc binding are not affected by significant structural rearrangement upon zinc binding supporting the idea that the binding site is ready to accommodate the metal. The interaction of SARS-CoV-2 Mpro with Zn2+ ion was also investigated by NMR. Moreover, zinc binding is able to inhibit protein activity, demonstrating that the zinc ion is capable of an efficient binding also in solution. These findings provide a solid ground for designing potent and selective inhibitors of SARS-CoV-2 Mpro suggesting that a zinc ion incorporated into suitable ligands interacting with additional sites at the protein surface can modulate the binding energy.
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.