2′-O methylation of RNA cap in SARS-CoV-2 captured by serial crystallography

Mateusz Wilamowski,George Minasov,Monica Rosas-Lemus,Ryan Chard,Youngchang Kim,Ludmilla Shuvalova,Nickolaus Saint,Darren A Sherrell,Karla J F Satchell,Robert Jedrzejczak,Ian T Foster,Natalia Maltseva,Karolina Michalska,Andrzej Joachimiak,Alex Lavens

doi:10.1073/pnas.2100170118

Abstract

The genome of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) coronavirus has a capping modification at the 5'-untranslated region (UTR) to prevent its degradation by host nucleases. These modifications are performed by the Nsp10/14 and Nsp10/16 heterodimers using S-adenosylmethionine as the methyl donor. Nsp10/16 heterodimer is responsible for the methylation at the ribose 2'-O position of the first nucleotide. To investigate the conformational changes of the complex during 2'-O methyltransferase activity, we used a fixed-target serial synchrotron crystallography method at room temperature. We determined crystal structures of Nsp10/16 with substrates and products that revealed the states before and after methylation, occurring within the crystals during the experiments. Here we report the crystal structure of Nsp10/16 in complex with Cap-1 analog (m7GpppAm2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

Highlights

There are three main pathogenic coronaviruses that cause major respiratory diseases in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1, 2]
The RNA capping involves several enzymatic steps performed by viral Nsps; Nsp13 is a bifunctional RNA/NTP triphosphatase and helicase that hydrolyses the first phosphate from the nascent RNA; an unknown guanylyltransferase transfers a guanosine monophosphate moiety to the pp-RNA forming GpppRNA. This is followed by transfer of a methyl group from the donor S-adenosylmethionine (AdoMet) to the guanidine N7 to form m7GpppA2′-OH-RNA (Cap-0-RNA) by Nsp10/14 heterodimer
While several structures of Nsp10/16 in a complex with Cap0 analog have been determined by single crystal cryocrystallography (Protein Data Bank [PDB] entries: 6WQ3, 6WRZ, 6WVN, 6WKS, 6YZ1, 6WKQ, 6WJT, 6W61, 6W75, 6W4H, 7BQ7, 7C2I, and 7C2J) [12, 15,16,17], none has been determined at room temperature

Summary

Introduction

There are three main pathogenic coronaviruses that cause major respiratory diseases in humans: severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [1, 2]. The RNA in the mature virion resembles human mRNA: 1) it is capped on its 5′-end, 2) it contains a 3′-poly-A tail, and 3) after infection, it can be directly translated to the two polyproteins Pp1a and Pp1ab using host machinery These polyproteins mature and are cut into 16 polypeptides and 15 nonstructural proteins (Nsps) that assemble into a large replication–transcription complex. We determined crystal structures of SARS-CoV-2 Nsp10/16 heterodimer in complex with substrates (Cap-0 analog and S-adenosyl methionine) and products (Cap-1 analog and S-adenosyl-L-homocysteine) at room temperature using synchrotron serial crystallography. Analysis of these structures will aid structure-based drug design against 2′-O-methyltransferase from SARS-CoV-2

Methods

Results

Conclusion