Role of the transmembrane domain in severe acute respiratory syndrome ( <scp>SARS</scp> ) coronavirus 2 spike for palmitoylation and membrane fusion

Palmitoylation is a reversible post-translational modification that enhances protein hydrophobicity and regulates cellular functions such as trafficking and signaling. In humans, this modification is catalyzed by 23 DHHC enzymes, but the mechanisms by which they recognize their substrates remain unclear. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein undergoes palmitoylation primarily by DHHC20 with subsequent modification by DHHC9 at 10 cytoplasmic tail (CT) cysteines, a modification crucial for membrane fusion and viral entry. Using AlphaFold2 modeling and site-directed mutagenesis, we identified three key components critical for efficient spike palmitoylation: (i) Lys1211 at the ectodomain-transmembrane domain (TMD) interface, likely facilitating electrostatic interactions with DHHC20's acidic residues; (ii) a stable trimeric TMD helix, where mutations at the trimer interface impair palmitoylation, in contrast to changes in outward-facing residues; and (iii) a conserved hydrophilic motif in the CT, located between acylated cysteine clusters, likely promoting optimal substrate positioning near DHHC20's catalytic site. Co-immunoprecipitation assays revealed that mutations in these residues disrupt spike-DHHC20 interactions, while leaving spike-DHHC9 binding unchanged, suggesting that they affect enzyme-substrate complex formation. Fusion assays revealed nuanced effects; while palmitoylation generally correlated positively with membrane fusion, certain exceptions highlighted the complex relationship between these processes. Mutations in the CT markedly reduce total spike palmitoylation but only modestly affect cell-cell fusion. Some substitutions in the TMD impair fusion with little change in overall acylation. Our findings elucidate the structural and biophysical determinants of spike palmitoylation and its distinct roles in membrane fusion, offering insights into SARS-CoV-2 pathogenesis and potential antiviral targets.