Fig. 4. A four-residue loop in coronavirus nsp16 alters the orientation of the RNA substrate.

(A) Wall-eyed stereo view of the superimposed capped RNAs mapped on the electrostatic potential surface of SARS-CoV-2 2′-O-MTase. RNAs are shown as sticks in bright pink (SARS-CoV-2), cyan (DENV NS5), yellow (VACV VP39) and green (hCMTr1). The nucleotides are labeled with numbers starting from m⁷G (position 0); 3* and 4* correspond to U₃ and A₄ in the Cap-1-RNA from SARS-CoV-2, 4’-6’ to A₄-A₅-A₆ in the capped mRNA from VACV. (B) Superimposition of the RNA binding sites of nsp16-nsp10 with Cap-0-RNA (SARS-CoV-2, magenta) and Cap-0 analog (MERS-CoV, blue). Protein chains are shown as ribbons in pink for SARS-CoV-2 (PDB 7JYY) and blue for MERS-CoV (PDB 5YNM) overlayed on the semitransparent solvent-exposed surface of nsp16 (beige) and nsp10 (teal). Direct hydrogen bond interactions of Asp⁶⁸⁷³ and Lys⁶⁸⁷⁴ with U₂ are shown as black, dashed lines. (C) Multiple sequence alignment of the loop region between β1 and αA from different coronaviruses, with Asp⁶⁸⁷³ and Lys⁶⁸⁷⁴ highlighted in red. SARS-CoV, severe acute respiratory syndrome; MERS-CoV, Middle East respiratory syndrome; MHV, murine hepatitis virus (MHV); HKU1, human coronavirus 1; H-CoV-229E, human coronavirus 229E; F-CoV, feline coronavirus; IBV, infectious bronchitis virus (IBV). (D and E) Structural and sequence alignments of nsp16 in pink, hCMTr1 in green, VP39 in yellow, and NS5 in cyan. Protein chains are shown as cartoon models with conserved Asp⁶⁸⁷³ and Lys⁶⁸⁷⁴ from nsp16 shown as sticks and the insertion loop highlighted in pink.