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 KD. 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.

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

“…A high-resolution Cryo-EM structure of a core fragment of Orf9b in complex with human TOM70 depicts the central part of Orf9b (amino acids 39-76) buried within the C-terminal domain of TOM70 [16]. Additionally, a crystal structure of a similar fragment of Orf9b (amino acids 43-78) in complex with human TOM70 was published recently and confirmed the localization of the core part of Orf9b within TOM70 [15]. Within the interaction interface of the Orf9b-TOM70 complex, both structures show a prominent amino acid of Orf9b, a serine at position 53 (S53), to form a hydrogen bond with a glutamate at position 477 (E477) of TOM70 in an overall hydrophobic region.…”

“…In previous studies, the complex formation of Orf9b and TOM70 was mainly characterized using purified proteins or synthetic peptides [15,16]. To investigate the interaction of both proteins under the conditions of a mammalian cytosol, we translated Orf9b in rabbit reticulocyte lysate (RL) in the presence of [ 35 S]-methionine (Figure 1A).…”

“…(H) Radiolabeled [ 35 A recent study identified two phosphorylation sites on Orf9b: a serine at position 50 (S50) and a serine at position 53 (S53) (Figure 1D) [17]. Orf9b S53 forms a prominent hydrogen bond with a conserved glutamate at position 477 in the C-terminal domain of TOM70 (E477) within the overall hydrophobic interaction interface of both proteins (Figure 1E) [15,16]. We hypothesized that the phosphorylation of Orf9b S53 would destabilize the interaction with TOM70 E477, which, in turn, could weaken the association of both proteins.…”

“…It has been speculated that inhibition of Hsp90 binding by Orf9b could be a possible reason for decreased IFN-I expression [13]. A study using isothermal titration calorimetry (ITC) indicated that the binding of an Orf9b-derived peptide inhibits the binding of an Hsp90-derived peptide to the N-terminal domain of TOM70, suggesting an allosteric inhibition [15]. However, the influence of Orf9b binding on the recognition of native Hsp90 was not investigated.…”

Coronavirus, the King Who Wanted More Than a Crown: From Common to the Highly Pathogenic SARS-CoV-2, Is the Key in the Accessory Genes?

“…The SARS-CoV-2 ORF9b protein was depicted to inhibit the type I IFN response through its interaction with the outer membrane mitochondrial adaptor TOM70 (Jiang et al, 2020b). Recently, the crystal structure of ORF9b in complex with the human TOM70 was resolved (Gao et al, 2021). Moreover, SARS-CoV-2 ORF9b antagonizes types I and III interferons by targeting multiple components of the RIG-I/MDA-5-MAVS, TLR3-TRIF, and cGAS-STING signaling pathways (Han et al, 2021).…”