Crystal structure of SARS-CoV-2 Orf9b in complex with human TOM70 suggests unusual virus-host interactions

Abstract

Although the accessory proteins are considered non-essential for coronavirus replication, accumulating evidences demonstrate they are critical to virus-host interaction and pathogenesis. Orf9b is a unique accessory protein of SARS-CoV-2 and SARS-CoV. It is implicated in immune evasion by targeting mitochondria, where it associates with the versatile adapter TOM70. Here, we determined the crystal structure of SARS-CoV-2 orf9b in complex with the cytosolic segment of human TOM70 to 2.2 Å. A central portion of orf9b occupies the deep pocket in the TOM70 C-terminal domain (CTD) and adopts a helical conformation strikingly different from the β-sheet-rich structure of the orf9b homodimer. Interactions between orf9b and TOM70 CTD are primarily hydrophobic and distinct from the electrostatic interaction between the heat shock protein 90 (Hsp90) EEVD motif and the TOM70 N-terminal domain (NTD). Using isothermal titration calorimetry (ITC), we demonstrated that the orf9b dimer does not bind TOM70, but a synthetic peptide harboring a segment of orf9b (denoted C-peptide) binds TOM70 with nanomolar K_D. While the interaction between C-peptide and TOM70 CTD is an endothermic process, the interaction between Hsp90 EEVD and TOM70 NTD is exothermic, which underscores the distinct binding mechanisms at NTD and CTD pockets. Strikingly, the binding affinity of Hsp90 EEVD motif to TOM70 NTD is reduced by ~29-fold when orf9b occupies the pocket of TOM70 CTD, supporting the hypothesis that orf9b allosterically inhibits the Hsp90/TOM70 interaction. Our findings shed light on the mechanism underlying SARS-CoV-2 orf9b mediated suppression of interferon responses.

Fig. 1. Expression and assembly of the hTOM70/SARS-CoV-2 orf9b complex.

a Diagrams of SARS-CoV-2 orf9b and hTOM70 proteins. TM transmembrane domain, NTD N-terminal domain, CTD C-terminal domain. b When co-expressed in E coli, three of hTOM70 truncations, hTOM70 ΔN1 (60-608), hTOM70 ΔN2 (106-608), and hTOM70 ΔN3 (235-608) without fusion tag co-elute with SARS-CoV-2 orf9b with an N-terminal His-tag. c SDS-PAGE analysis of fractions eluted from gel filtration column, demonstrating hTOM70 ΔN2 and SARS-CoV-2 orf9b formed stable complex. d Left, size-exclusion chromatography and right, analytical ultracentrifugation analyses three samples: co-expressed SARS-CoV-2 orf9b/hTOM70 ΔN2 complex (red), alone expressed hTOM70 (green) and SARS-CoV-2 orf9b (homodimeric, black). The calculated molecular weight is indicated on top of the peaks. Data for SARS-CoV-2 orf9b/hTOM70 ΔN2 complex are indicated with stars.

Fig. 2. Crystal structure of the hTOM70/SARS-CoV-2 orf9b complex.

a Left, ribbon model of the hTOM70 cytosolic domain with SARS-CoV-2 orf9b occupying the CTD pocket. The NTD of hTOM70 is colored blue, the CTD is cyan and orf9b is magenta. Tetratricopeptide repeat (TPR) motifs and secondary structure elements are indicated. Right, magnified view of SARS-CoV-2 orf9b inside the CTD pocket. The omit map (black mesh, calculated by program phenix.composite_omit_map) for orf9b is superimposed on the model. b Ribbon models of dimeric SARS-CoV-2 orf9b and the central portion of the same protein when bound to hTOM70. The segment of SARS-CoV-2 orf9b 43-78 adopts a β-strand rich structure in its dimeric form (right) and an α-helix rich structure in its bound form (left). c–e Details of polar interactions between SARS-CoV-2 orf9b and hTOM70.

Fig. 3. Orf9b binding induced conformational rearrangement.

a Superimposition of the hTOM70/SARS-CoV-2 orf9b complex with yeast TOM70 (PDB id: 2GW1). The NTD of hTOM70 is colored blue, the CTD is colored cyan. SARS-CoV-2 orf9b is colored magenta. b Superimposition of the hTOM70/SARS-CoV-2 orf9b complex with yeast TOM71 (PDB id: 3FP3). hTOM70 and SARS-CoV-2 orf9b are colored as in panel a. c Superimposition of the hTOM70/SARS-CoV-2 orf9b complex with yeast TOM70 and TOM71. Only the bridging helices and the bottom helices are shown to illustrate conformational rearrangement induced by SARS-CoV-2 orf9b binding.

Fig. 4. Cooperative binding at the NTD- and CTD-pockets.

a Surface electrostatic potential plot of hTOM70 with SARS-CoV-2 orf9b accommodating the CTD-pocket shown in magenta cartoon representation. b Superimposition of hTOM70 (blue) with the yeast TOM71-EEVD complex (PDB id: 3FP2). Yeast TOM71 is colored gray and the EEVD motif is colored yellow. c The EEVD motif (yellow) is modeled in the NTD-pocket of hTOM70. d Left, binding isotherms for the interaction of hTOM70 and the C-peptide (derived from the visible portion of SARS-CoV-2 orf9b in the crystal structure), which indicates an endothermic process; right, binding isotherms for pre-formed hTOM70/SARS-CoV-2 orf9b complex and the C-peptide, which shows little nor no binding. e Left, binding isotherms for the interaction of hTOM70 and the N-peptide (harboring the EEVD motif at the C-terminal region of Hsp90), which indicates an exothermic process; right, binding isotherms for pre-formed hTOM70/SARS-CoV-2 orf9b complex and the N-peptide; the binding affinity is reduced significantly.