The sequence and structural integrity of the SARS-CoV-2 Spike protein transmembrane domain is crucial for viral entry

The Spike protein of SARS-CoV-2 is a type I membrane protein that mediates target cell recognition and membrane fusion. Its transmembrane domain (TMD) is usually considered a passive physical anchor that attaches the protein to the viral envelope. However, current knowledge of the dynamics and biogenesis of integral membrane proteins indicates that TMDs generally play key functions beyond membrane anchoring. Here, we explored the role of the SARS-CoV-2 Spike protein TMD during viral entry. To do so, we generated a series of amino acid substitutions and insertions within the hydrophobic core of the TMD and analyzed their impact on protein function. Our results indicate that the SARS-CoV-2 Spike protein is highly susceptible to changes in its TMD. Furthermore, the TMD sequence and structural parameters relevant for viral entry are distributed across the entire segment, albeit with a stronger implication of the N-terminal portion. The relative orientation of the regions before and after the TMD was also found to be relevant for particle entry. Finally, our results suggest that the SARS-CoV-2 Spike protein TMD can form homo-oligomers through a motif in which small residues are necessary.