Belda, D. | bioRxiv Science

bioRxiv2026

Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase

Protein N-glycosylation is catalysed by the oligosaccharyltransferase (OST) complex, yet how local sequon composition controls substrate selection by human OST remains poorly understood. Here we combine atomistic molecular dynamics, alchemical free-energy calculations, in cellulo glycosylation assays and human glycoproteome analysis to define the molecular basis of sequence-dependent N-glycosylation. We built, to our knowledge, the first atomistic model of human OST with both the lipid-linked oligosaccharide donor and an acceptor peptide simultaneously accommodated in a catalytically competent complex. Simulations show that conserved STT3 motifs stabilize the acceptor Asn and explain how substitutions at positions 0, +1 and +2 alter binding and productive geometry. Cellular assays in HEK 293T cells validate the predicted loss of glycosylation for N0Q sequon, +1 Pro and the +2 hierarchy Thr > Ser >> Cys. Analysis of 14,800 human glycosites confirms these rules at proteome scale.

biochemistry↗

bioRxiv2024

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

The Spike (S) protein of SARS-CoV-2 is a type I membrane protein that mediates target cell recognition and membrane fusion. While its transmembrane domain (TMD) is traditionally viewed as a passive anchor to the viral envelope, emerging evidence suggests that TMDs often play active roles in the biogenesis and function of membrane proteins. Here, we investigated the functional role of the SARS-CoV-2 S protein TMD during viral entry. To this end, we introduced a series of amino acid substitutions and insertions within the hydrophobic core of the TMD and assessed their impact on S protein activity. Our findings reveal that the SARS-CoV-2 S protein is susceptible to alterations in its TMD. Functional determinants, including sequence features and structural parameters critical for viral entry, are distributed throughout the TMD, with a more pronounced contribution from its N-terminal region. We also demonstrate that the relative orientation of the regions flanking the TMD influences viral entry. Finally, our data suggest that the TMD mediates homo-oligomerization through a motif enriched in small residues, underscoring its functional importance beyond membrane anchoring.

molecular biology↗