Biology subjects

Tropea, B.

Publications and source records attributed to Tropea, B..

3 recordsLinked to original sources

bioRxiv2026

How the terminal glucoside of the N-glycan donor affects the catalytic efficiency of the eukaryotic oligosaccharyltransferase

The eukaryotic oligosaccharyltransferase (OST) is the enzyme responsible for initiating N-glycosylation of secreted proteins by transferring a pre-assembled lipid-linked oligosaccharide (LLO) donor to target asparagine residues most often found within N-x-S/T consensus sequences, or sequons. OST preferentially selects LLO donors with a distinctive glucoside Glc-(1-2)-Glc-(1-3)-Glc-(1-3)-capping the A-branch. After the N-glycosylation reaction, this motif is cleaved in a stepwise manner from the immature N-glycan structure before the folded glycoprotein exits the endoplasmic reticulum quality control (ERQC) cycle. While the -Glc-(1-3)-Glc-(1-3)-disaccharide is an important flag regulating binding to the calreticulin/calnexin chaperones, the terminal Glc-(1-2)-is removed immediately after OST catalysis, suggesting that its biological function may be directly linked to the OST catalytic efficiency. To understand how and why this capping motif affects the OST N-glycosylation efficiency, we rebuilt 3D models of the yeast OST in complex with an acceptor peptide and LLO donors substrates with and without terminal Glc-(1-2)-, and analysed their stability and dynamics with all-atom molecular dynamics (MD) simulations through both conventional, and Gaussian-accelerated (GaMD) sampling schemes. Our results indicate that the terminal Glc-(1-2)-is essential to anchor the full-length LLO donor to the OST through a complex network of intermolecular contacts extending from the catalytic site to distal subdomains. We show how this contact network is crucial to preserve the LLO catalytically productive alignment of its reducing end. We also show that the removal of the terminal Glc-(1-2)-leads to an increased flexibility of the LLO, which displaces the reducing end and redistributes the conformational ensemble towards misaligned states, which are less catalytically productive. These results provide a mechanistic basis linking the catalytic efficiency of the eukaryotic OST to the distinctive glucosylated structure of the LLO donor.

biophysics↗

bioRxiv2025

Regulation of N-glycosylation efficiency by eukaryotic oligosaccharyltransferase

Oligosaccharyltransferase (OST) catalyses the key step of N-glycosylation, transferring immature N-glycans to select Asn residues in nascent proteins in the endoplasmic reticulum (ER). Asn are more likely to be glycosylated in the context of a "glycosylation sequon", N-x-S/T (x=P), but not every sequon is glycosylated. Tight positive and negative regulation of site-specific N-glycosylation across the glycoproteome is essential because some Asn require glycosylation for productive protein folding or function, while others must remain unglycosylated. Despite its importance, the underlying bases for this regulation are illdefined. Here we characterise the molecular determinants regulating OST catalytic efficiency. We identify preferred substrates of OST, and show that the local sequence determines glycosylation efficiency by fine-tuning binding to an extended groove in OST along an eight residue stretch centered at the Asn acceptor. Tight control of OST activity is achieved through a switch, set ON when a peptide binds with high complementarity to the groove, triggering release of the assisting base Glu350 for catalysis. Based on analysis of buried sequons, we identify sequence characteristics associated with inherently non-preferred acceptor substrates for OST, and show these have a high propensity for local secondary structure incompatible with the optimal conformation of a peptide bound to OST. Finally, we validate these mechanisms by examining the determinants of efficient glycosylation at a variably glycosylated Asn in a set of human Fabs. These mechanistic and functional insights show how local protein sequence controls glycosylation occupancy with immediate implications in secretory protein function and evolution, pathogen immunity and epistasis, and in engineering protein sequences for the desired degree of site-specific glycosylation.

biochemistry↗

bioRxiv2023

Restoring Protein Glycosylation with GlycoShape

During the past few years, we have been witnessing a revolution in structural biology. Leveraging on technological and computational advances, scientists can now resolve biomolecular structures at the atomistic level of detail by cryogenic electron microscopy (cryo-EM) and predict 3D structures from sequence alone by machine learning (ML). One technique often supports the other to provide the view of atoms in molecules required to capture the function of molecular machines. An example of the extraordinary impact of these advances on scientific discovery and on public health is given by how structural information supported the rapid development of COVID-19 vaccines based on the SARS-CoV-2 spike (S) glycoprotein. Yet, none of these new technologies can capture the details of the dense coat of glycans covering S, which is responsible for its natural, biologically active structure and function and ultimately for viral evasion. Indeed, glycosylation, the most abundant post-translational modification of proteins, is largely invisible through experimental structural biology and in turn it cannot be reproduced by ML, because of the lack of data to learn from. Molecular simulations through high-performance computing (HPC) can fill this crucial information gap, yet the computational resources, the users skills and the long timescales involved limit applications of molecular modelling to single study cases. To broaden access to structural information on glycans, here we introduce GlycoShape (https://glycoshape.org) an open access (OA) glycan structure database and toolbox designed to restore glycoproteins to their native functional form by supplementing the structural information available on proteins in public repositories, such as the RCSB PDB (www.rcsb.org) and AlphaFold Protein Structure Database (https://alphafold.ebi.ac.uk/), with the missing glycans derived from over 1 ms of cumulative sampling from molecular dynamics (MD) simulations. The GlycoShape Glycan Database (GDB) currently counts over 435 unique glycans principally covering the human glycome and with additional structures, fragments, and epitopes from other eukaryotic and prokaryotic organisms. The GDB feeds into Re-Glyco, a bespoke algorithm in GlycoShape designed to rapidly restore the natural glycosylation to protein 3D structures and to predict N-glycosylation occupancy, where unknown. Ultimately, integration of GlycoShape with other OA protein structure databases can provide a step-change in scientific discovery, from the structural and functional characterization of the active form of biomolecules, all the way down to pharmacological applications and drug discovery.

biophysics↗