Biology subjects

Harbison, A. M.

Publications and source records attributed to Harbison, A. M..

8 recordsLinked to original sources

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↗

bioRxiv2023

GotGlycans: Role of N343 Glycosylation on the SARS-CoV-2 S RBD Structure and Co-Receptor Binding Across Variants of Concern

Glycosylation of the SARS-CoV-2 spike (S) protein represents a key target for viral evolution because it affects both viral evasion and fitness. Successful variations in the glycan shield are difficult to achieve though, as protein glycosylation is also critical to folding and to structural stability. Within this framework, the identification of glycosylation sites that are structurally dispensable can provide insight into the evolutionary mechanisms of the shield and inform immune surveillance. In this work we show through over 45 s of cumulative sampling from conventional and enhanced molecular dynamics (MD) simulations, how the structure of the immunodominant S receptor binding domain (RBD) is regulated by N-glycosylation at N343 and how this glycans structural role changes from WHu-1, alpha (B.1.1.7), and beta (B.1.351), to the delta (B.1.617.2) and omicron (BA.1 and BA.2.86) variants. More specifically, we find that the amphipathic nature of the N-glycan is instrumental to preserve the structural integrity of the RBD hydrophobic core and that loss of glycosylation at N343 triggers a specific and consistent conformational change. We show how this change allosterically regulates the conformation of the receptor binding motif (RBM) in the WHu-1, alpha and beta RBDs, but not in the delta and omicron variants, due to mutations that reinforce the RBD architecture. In support of these findings, we show that the binding of the RBD to monosialylated ganglioside co-receptors is highly dependent on N343 glycosylation in the WHu-1, but not in the delta RBD, and that affinity changes significantly across VoCs. Ultimately, the molecular and functional insight we provide in this work reinforces our understanding of the role of glycosylation in protein structure and function and it also allows us to identify the structural constraints within which the glycosylation site at N343 can become a hotspot for mutations in the SARS-CoV-2 S glycan shield.

biophysics↗

bioRxiv2021

Fine-tuning the Spike: Role of the nature and topology of the glycan shield structure and dynamics of SARS-CoV-2 S

The dense glycan shield is an essential feature of the SARS-CoV-2 spike (S) architecture, key to immune evasion and to the activation of the prefusion conformation. Recent studies indicate that the occupancy and structures of the SARS-CoV-2 S glycans depend not only on the nature of the host cell, but also on the structural stability of the trimer; a point that raises important questions about the relative competence of different glycoforms. Moreover, the functional role of the glycan shield in the SARS-CoV-2 pathogenesis suggests that the evolution of the sites of glycosylation is potentially intertwined with the evolution of the protein sequence to affect optimal activity. Our results from multi-microsecond molecular dynamics simulations indicate that the type of glycosylation at N234, N165 and N343 greatly affects the stability of the receptor binding domain (RBD) open conformation, and thus its exposure and accessibility. Furthermore, our results suggest that the loss of glycosylation at N370, a newly acquired modification in the SARS-CoV-2 S glycan shields topology, may have contributed to increase the SARS-CoV-2 infectivity as we find that N-glycosylation at N370 stabilizes the closed RBD conformation by binding a specific cleft on the RBD surface. We discuss how the absence of the N370 glycan in the SARS-CoV-2 S frees the RBD glycan binding cleft, which becomes available to bind cell-surface glycans, potentially increases host cell surface localization.

biophysics↗

bioRxiv2020

Citizen Scientists Create an Exascale Computer to Combat COVID-19

SARS-CoV-2 has intricate mechanisms for initiating infection, immune evasion/suppression, and replication, which depend on the structure and dynamics of its constituent proteins. Many protein structures have been solved, but far less is known about their relevant conformational changes. To address this challenge, over a million citizen scientists banded together through the Folding@home distributed computing project to create the first exascale computer and simulate an unprecedented 0.1 seconds of the viral proteome. Our simulations capture dramatic opening of the apo Spike complex, far beyond that seen experimentally, which explains and successfully predicts the existence of cryptic epitopes. Different Spike homologues modulate the probabilities of open versus closed structures, balancing receptor binding and immune evasion. We also observe dramatic conformational changes across the proteome, which reveal over 50 cryptic pockets that expand targeting options for the design of antivirals. All data and models are freely available online, providing a quantitative structural atlas.

biophysics↗

bioRxiv2020

Shielding and Beyond: The Roles of Glycans in SARS-CoV-2 Spike Protein

The ongoing COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in more than 15,000,000 infections and 600,000 deaths worldwide to date. Antibody development efforts mainly revolve around the extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates the host cell entry by binding to the angiotensin-converting enzyme 2 (ACE2). Similar to many other viruses, the SARS-CoV-2 spike utilizes a glycan shield to thwart the host immune response. Here, we built a full-length model of glycosylated SARS-CoV-2 S protein, both in the open and closed states, augmenting the available structural and biological data. Multiple microsecond-long, all-atom molecular dynamics simulations were used to provide an atomistic perspective on the roles of glycans, and the protein structure and dynamics. We reveal an essential structural role of N-glycans at sites N165 and N234 in modulating the conformational dynamics of the spikes receptor binding domain (RBD), which is responsible for ACE2 recognition. This finding is corroborated by biolayer interferometry experiments, which show that deletion of these glycans through N165A and N234A mutations significantly reduces binding to ACE2 as a result of the RBD conformational shift towards the "down" state. Additionally, end-to-end accessibility analyses outline a complete overview of the vulnerabilities of the glycan shield of SARS-CoV-2 S protein, which may be exploited by therapeutic efforts targeting this molecular machine. Overall, this work presents hitherto unseen functional and structural insights into the SARS-CoV-2 S protein and its glycan coat, providing a strategy to control the conformational plasticity of the RBD that could be harnessed for vaccine development.

biophysics↗

bioRxiv2020

How and why plants and humans N-glycans are different: Insight from molecular dynamics into the "glycoblocks" architecture of complex carbohydrates

N-glycosylation is one of the most abundant and diverse post-translational modifications of proteins, implicated in protein folding and structural stability, and mediating interactions with receptors and with the environment. All N-glycans share a common core from which linear or branched arms stem from, with functionalization specific to different species and to the cells health and disease state. This diversity generates a rich collection of structures, all diversely able to trigger molecular cascades and to activate pathways, which also include adverse immunogenic responses. These events are inherently linked to the N-glycans 3D architecture and dynamics, which remain for the large part unresolved and undetected because of their intrinsic structural disorder. In this work we use molecular dynamics (MD) simulations to provide insight into N-glycans 3D structure by analysing the effects of a set of very specific modifications found in plants and invertebrate N-glycans, which are immunogenic in humans. We also compare these structural motifs and combine them with mammalian N-glycans motifs to devise strategies for the control of the N-glycan 3D structure through sequence. Our results suggest that the N-glycans architecture can be described in terms of the local spatial environment of groups of monosaccharides. We define these "glycoblocks" as self-contained 3D units, uniquely identified by the nature of the residues they comprise, their linkages and structural/dynamic features. This alternative description of glycans 3D architecture can potentially lead to an easier prediction of sequence-to-structure relationships in complex carbohydrates, with important implications in glycoengineering design.

biophysics↗

bioRxiv2019

An atomistic perspective on ADCC quenching by core-fucosylation of IgG1 Fc N-glycans from enhanced sampling molecular dynamics

The immunoglobulin type G (IgG) Fc N-glycans are known to modulate the interaction with membrane-bound Fc {gamma} receptors (Fc{gamma}Rs), fine-tuning the antibodys effector function in a sequence-dependent manner. Particularly interesting in this respect are the roles of galactosylation, which levels are linked to autoimmune conditions and aging, of core fucosylation, which is known to reduce significantly the antibody-dependent cellular cytotoxicity (ADCC), and of sialylation, which also reduces ADCC but only in the context of core-fucosylation. In this work we provide an atomistic level perspective through enhanced sampling computer simulations, based on replica exchange molecular dynamics (REMD), to understand the molecular determinants linking the Fc N-glycans sequence to the observed IgG1 function. Our results indicate that the two symmetrically opposed N-glycans interact extensively through their core trimannose residues. At room temperature the terminal galactose on the (1-6) arm is restrained to the protein through a network of interactions that keep the arm outstretched, meanwhile the (1-3) arm extends towards the solvent where a terminal sialic acid remains fully accessible. We also find that the presence of core fucose interferes with the extended sialylated (1-3) arm, altering its conformational propensity and as a consequence of steric hindrance, significantly enhancing the Fc dynamics. Furthermore, structural analysis shows that the core fucose position within the Fc core obstructs the access of N162 glycosylated Fc{gamma}Rs very much like a \"door-stop\", potentially decreasing the IgG/Fc{gamma}R binding free energy. All of these factors could represent important clues to understand at the molecular level the dramatic reduction of ADCC as a result of core fucosylation and provide an atomistic level-of-detail framework for the design of high potency IgG1 Fc N-glycoforms.

biophysics↗

bioRxiv2018

Sequence-to-structure dependence of isolated IgG Fc complex biantennary N-glycans: A molecular dynamics study

Fc glycosylation of human immunoglobulins G (IgGs) is essential for their structural integrity and activity. Interestingly, the specific nature of the Fc glycoforms is known to modulate the IgG effector function. Indeed, while core-fucosylation of IgG Fc-glycans greatly affects the antibody-dependent cell-mediated cytotoxicity (ADCC) function, with obvious repercussions in the design of therapeutic antibodies, sialylation can reverse the antibody inflammatory response, and galactosylation levels have been linked to aging, to the onset of inflammation, and to the predisposition to rheumatoid arthritis. Within the framework of a structure-to-function relationship, we have studied the role of the N-glycan sequence on its intrinsic conformational propensity. Here we report the results of a systematic study, based on extensive molecular dynamics (MD) simulations in excess of 62 {micro}s of cumulative simulation time, on the effect of sequence on the structure and dynamics of increasingly larger, complex biantennary N-glycoforms, i.e. from chitobiose to the larger N-glycan species commonly found in the Fc region of human IgGs. Our results show that while core fucosylation and sialylation do not affect the intrinsic dynamics of the isolated (unbound) N-glycans, galactosylation of the (1-6) arm shifts dramatically its conformational equilibrium from an outstretched to a folded conformation. These findings are in agreement with and can help rationalize recent experimental evidence showing a differential recognition of positional isomers in glycan array data and also the preference of sialyltransferase for the more reachable, outstretched (1-3) arm in both isolated and Fc-bound N-glycans.

biophysics↗