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

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 (^m7GpppA_m2'-O). Inhibition of Nsp16 activity may reduce viral proliferation, making this protein an attractive drug target.

Keywords: CAP-1; Nsp10/16; SARS-CoV-2; mRNA; serial crystallography.

Fig. 1.

Fixed-target serial crystallography data collection on Nsp10/16 crystals from SARS-CoV-2. System for SSX with moving crystal holder developed at SBC beamline 19ID.

Fig. 2.

Crystal structure of the Nsp10/16 in a complex with ^m7GpppA_m2′-O (Cap-1) and AdoHcys determined using fixed-target SSX at 295 K. (A) Diagram of the 2′-O methyl transfer reaction performed by Nsp10/16; methyl groups are highlighted with blue ovals. (B) The assembly of the Nsp10/16 heterodimer; Nsp10 (pink), Nsp16 (yellow) in complex with Cap-1 (aquamarine), and AdoHcys (green) (PDB entry 7JHE). (C) The ^m7Gppp at the potential allosteric site of Nsp16. (D) The Coulombic electrostatic potential mapped on the surface of the Nsp10/16 heterodimer in complex with Cap-1/AdoHcys at 295 K calculated using default settings in UCSF Chimera. (E) 2mFo-DFc composite omit map calculated for ^m7Gpp and contoured at 1.2 σ (PDB entry 7JPE). The Nsp16 is shown as yellow ribbon, selected residues (yellow), and ^m7Gpp are depicted as pink sticks.

Fig. 3.

Comparison of crystal structures of the Nsp10/16 determined at 295 K and at 100 K. (A) Crystal structure of the Nsp10/16 with Cap-1 and AdoHcys (yellow, 7JHE) determined using SSX superimposed with Nsp10/16 with Cap-0 and AdoHcys (red, 6WQ3) determined at 100 K. (B) Comparison of 295 K crystal structures of Nsp10/16/Cap-1/AdoHcys (yellow, 7JHE) superimposed to the structure of Nsp10/16/Cap-0/AdoMet (black, 7JPE). (C) Comparison of 295 K crystal structures of the Nsp10/16/Cap-1/AdoHcys (yellow, 7JHE) superimposed to the structure of Nsp10/16/AdoMet (pink, 6XKM). (D) Comparison of the Nsp10/16 crystal structures with AdoMet determined respectively using SSX (pink, 6XKM) and standard data collection at 100 K (green, 6W4H). (E and F) Binding of Cap-1 to Nsp16. Plots of rmsd (E) and B factor of structures with AdoMet and Cap-1/AdoHcys (F).

Fig. 4.

Methylation of the 2′-O-ribose of the ^m7GpppA catalyzed by Nsp10/16 from SARS-CoV-2. (A) Active site of Nsp16 2′-O methyltransferase (yellow) in complex with ^m7GpppA_m2′-O (Cap-1) as aquamarine sticks and AdoHcys as green sticks. (B) Magnification of the active site depicts catalytic tetrad Lys46-Asp130-Lys170-Glu203 essential for the 2′-O MTase activity shown as yellow sticks. Red dashed lines show close distances between residues and arrows depict simplified nucleophilic attack and subsequent movement of methyl group to the 2′-O position. The water molecule is represented as a blue sphere. (C) The 2mFo-DFc maps contoured at 1.2 σ around ligands of the structures determined by SSX; cap analogs are in blue, AdoHcys in pink, and AdoMet in green.