An extended motif in the SARS-CoV-2 spike modulates binding and release of host coatomer in retrograde trafficking

Abstract

Abstractβ-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify an extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

Cytoplasmic tail determines the membrane trafficking and localization of SARS-CoV-2 spike protein

“…None of the coronavirus S proteins contained a C-terminal acidic residue within the K-X-H-X-X, K-X-K-X-X, or K-X-R-X X dibasic motif. This result was consistent with the former result focusing on the binding motif rather than the acid-base property and complementary charged function ( Dey et al, 2022 ).…”

“…The cargoes of COPI are numerous, such as escaped proteins or lipids. Interestingly, COPI-coated vesicles are involved in the life cycling of multiple viruses ( Zhang et al, 2009 ; Yang and Zhang, 2012 ; Li et al, 2014 ; Zhang and Zhang, 2017 ), including SARS-CoV-2 ( Cattin-Ortolá et al, 2021 ; Jennings et al, 2021 ; Dey et al, 2022 ).…”

“…Researchers have examined the effect of S protein C-terminal residues in modulating COPI binding using a biolayer interferometry assay (BLI) assay ( Dey et al, 2022 ). Three diverse mutations of the S protein hepta-peptide, namely acidic (Glu), basic (Arg), and neutral (Gln) amino acid, were employed to test the binding effects that were influenced by the acid-base property of C-terminal residues.…”

“…According to the in silico modeling of S protein hepta-peptide, αCOPI-WD40 domain contained predominantly polar residues. Among these residues, Arg57, Asp115, and Tyr139 played significant roles in the stabilization of S protein hepta-peptide binding ( Dey et al, 2022 ). Arg57 in αCOPI-WD40 interacted with His1271, Tyr1272, and Thr1273 in S. Asp115 in αCOPI-WD40 associated with Lys1269 side-chain of S. Moreover, this side-chain of Lys1269 further interacted with the hydroxyl oxygen in Tyr139 side-chain of αCOPI-WD40 domain.…”

“…To further confirm the function of amino acid residues in αCOPI-WD40 domain, experiments on the mutagenesis of these three αCOPI-WD40 residues to Ala were conducted, aiming to detect the binding efficiency ( Dey et al, 2022 ). These three mutants were analyzed for binding to the wild-type S protein hepta-peptide.…”