Structural basis for polyuridine tract recognition by SARS-CoV-2 Nsp15

Figures

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  Fig. 1.. CryoEM reconstructions of apo- and RNA-bound SARS-CoV-2 Nsp15. (A) Schematic representation of the domain organization and construct design of Nsp15 (top). dsRNA substrate sequence used for the reconstitution of Nsp15-RNA complex (bottom). (B) 2D class averages of the reconstituted Nsp15-RNA complex. A subset of the 2D classes shows extra density stemming from the core of the 2D density (indicated by arrows). (C) Two orthogonal views of the cryoEM reconstruction of apo-Nsp15 at 2.3 Å resolution. (D) Two orthogonal views of the cryoEM reconstruction of the Nsp15-RNA complex at 2.7 Å resolution. RNA density is shown in two different isosurface threshold levels (grey and red surfaces) to show both high-resolution and low-resolution features.

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  Fig. 2.. Two distinct states of Nsp15-RNA complex. (A) CryoEM density maps (top) and the corresponding atomic models (bottom) of the Nsp15-RNA state 1 (left) and state 2 (right) structures. The RNA chains in green and red correspond to positive and negative strands of the coronavirus genome, respectively. (B) Overlay of the RNA models from the two states on the consensus Nsp15 structure in the ribbon model (left) and surface model (right). The dotted line indicates the trajectory of the bound dsRNA.

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  Fig. 3.. Poly(U) uracil base flipping at nuclease active site center. (A) CryoEM density and the corresponding atomic model of the RNA bound to Nsp15. Two orthogonal views of the bound dsRNA density and its model (left). Density fitting of the RNA model of the U0-U+3:A0-A+3 (middle). Density fitting of the RNA model of U−3-U0:A+3-A0 (right). (B) Structure of the endonuclease active site center and recognition mechanism of flipped scissile U0 base. (C) Surface electrostatic potential of the Nsp15 around the endonuclease active site center and the interactions with the target U0 with both 3′- and 5′-phosphates (depicted in sticks). The surface area is colored according to the calculated electrostatic potential from −10.0 kT/e (red) to +10.0 kT/e (blue). (D) Recognition of the open major groove of the dsRNA substrate by Nsp15. W332 is intercalated into the space that would have been occupied by the flipped U0 base within the RNA duplex. M330 and Y342 additionally create the hydrophobic surface to facilitate the major groove interaction. Two polar residues K334 and E339 interact with U+2 and U+1, respectively. (E) The key hydrophobic residues responsible for base-flipping. The side chain of…

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  Fig. 4.. Bound RNA forms extensive interactions with Nsp15 hexamer across three subunits. (A) Interface between a patch of -314VSKV317- of subunit A’s CTD and A0 and A+1 of the positive strand. (B) Interface between a patch of -242HSQ244- of subunit A’s CTD and A−8 and A−7 of the positive strand and U+1 of the negative strand. (C) Interface between subunit B’s MD and U+10 of the negative strand and A−8, A−6 of the positive strand. (D) Interface between subunit B’s NTD and the U−4-U−1 of the negative strand. (E) Interface between subunit D’s MD and U−14-U−12 of the negative strand and A+16 of the positive strand.