Structural basis for the multimerization of nonstructural protein nsp9 from SARS-CoV-2

Fig. 1. Overall structure of the SARS-CoV-2 nsp9 protomer. a Different views of the protomeric architecture of SARS-CoV-2 nsp9 with the secondary structure labeled. The α-helix, β-sheets, and loops are colored in green, orange, and white, respectively. b–f Superimposition of SARS-CoV-2 nsp9 with other nsp9 structures in the coronavirus family. The nsp9s from SARS-CoV-2, SARS-CoV, avian infectious bronchitis virus (IBV), human coronavirus 229E (hCoV-229E), porcine delta coronavirus (PDCoV), and porcine epidemic diarrhea virus (PDEV) are colored in orange, light blue, marine, white, and salmon, respectively. The PDB codes are indicated in the lower right corner. gSequence alignment of CoV nsp9 homologs. Comparison of the SARS-CoV, IBV, HCoV, PDCoV, and PDEV sequences with the SARS-CoV-2 sequence. Identical residues are highlighted in red, and conserved residues are shown in yellow. The table was produced with ESPript 3.0, using secondary structure elements for SARS-CoV-2 nsp9 assigned using DSSP. Residues boxed in red are completely conserved

Fig. 2. Structure of the SARS-CoV-2 nsp9 tetramer. a Architecture and assembly of the nsp9 tetramer. Molecules a, b, c, and d in the stable tetramer are colored in orange, light blue, bright orange and pale green, respectively, and depicted in cartoon representations. b The stable tetramer is colored, labeled as Fig. 2a and depicted in cartoon and surface representations. c The electrostatic potential surface of the stable tetramer. Blue, positive charge (+ 70 kBT); Red, negative charge (− 70 kBT)

Fig. 3. Details of structure of the SARS-CoV-2 nsp9 tetramer and function in nucleic acid binding. a, b Details of Ic/d and Ia/b in the helix interface. The molecules in these two interfaces are shown as cartoons and colored and labeled as in Fig. 2a. The expanded boxed area to the right shows a detailed view of the critical residues in the Ic/d and Ia/b interfaces, which are shown as sticks. c Molecules involved in Ib/c. The molecules in this interface are shown as cartoons and colored and labeled as in Fig. 2a. d–f Detailed views of the first, second, and third contact regions in interface Ib/c, respectively. The secondary structures of each contact region are colored and labeled according to Fig. 2a and the critical residues in each region are shown as sticks. g Single-stranded DNA (ssDNA)-binding abilities of SARS-CoV-2 nsp9, as determined by electrophoretic mobility shift assay. h Single-stranded RNA (ssRNA)-binding abilities of SARS-CoV-2 nsp9. The length of ssDNA, the state of SARS-CoV-2 nsp9, and the concentration of proteins used in the reaction system are indicated above the gel