Search

Despite ground-breaking innovations in experimental structural biology and protein structure prediction techniques, capturing the structure of the glycans that functionalize proteins remains a challenge. Here we introduce GlycoShape ( https://glycoshape.org ), an open-access glycan structure database and toolbox designed to restore glycoproteins to their native and functional form in seconds. The GlycoShape database counts over 500 unique glycans so far, covering the human glycome and augmented by elements from a wide range of organisms, obtained from 1 ms of cumulative sampling from molecular dynamics simulations. These structures can be linked to proteins with a robust algorithm named Re-Glyco, directly compatible with structural data in open-access repositories, such as the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) and AlphaFold Protein Structure Database, or own. The quality, performance and broad applicability of GlycoShape is demonstrated by its ability to predict N-glycosylation occupancy, scoring a 93% agreement with experiment, based on screening all proteins in the PDB with a corresponding glycoproteomics profile, for a total of 4,259 N-glycosylation sequons., (© 2024. The Author(s).)

A key capability of ion channels is the facilitation of selective permeation of certain ionic species across cellular membranes at high rates. Due to their physiological significance, ion channels are of great pharmaceutical interest as drug targets. The polymodal signal-detecting transient receptor potential (TRP) superfamily of ion channels forms a particularly promising group of drug targets. While most members of this family permeate a broad range of cations including Ca 2+ , TRPM4 and TRPM5 are unique due to their strong monovalent selectivity and impermeability for divalent cations. Here, we investigated the mechanistic basis for their unique monovalent selectivity by in silico electrophysiology simulations of TRPM5. Our simulations reveal an unusual mechanism of cation selectivity, which is underpinned by the function of the central channel cavity alongside the selectivity filter. Our results suggest that a subtle hydrophobic barrier at the cavity entrance ("hydrophobic funnel") enables monovalent but not divalent cations to pass and occupy the cavity at physiologically relevant membrane voltages. Monovalent cations then permeate efficiently by a cooperative, distant knock-on mechanism between two binding regions in the extracellular pore vestibule and the central cavity. By contrast, divalent cations do not enter or interact favorably with the channel cavity due to its raised hydrophobicity. Hydrophilic mutations in the transition zone between the selectivity filter and the central channel cavity abolish the barrier for divalent cations, enabling both monovalent and divalent cations to traverse TRPM5., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Glycans constitute the most complicated post-translational modification, modulating protein activity in health and disease. However, structural annotation from tandem mass spectrometry (MS/MS) data is a bottleneck in glycomics, preventing high-throughput endeavors and relegating glycomics to a few experts. Trained on a newly curated set of 500,000 annotated MS/MS spectra, here we present CandyCrunch, a dilated residual neural network predicting glycan structure from raw liquid chromatography-MS/MS data in seconds (top-1 accuracy: 90.3%). We developed an open-access Python-based workflow of raw data conversion and prediction, followed by automated curation and fragment annotation, with predictions recapitulating and extending expert annotation. We demonstrate that this can be used for de novo annotation, diagnostic fragment identification and high-throughput glycomics. For maximum impact, this entire pipeline is tightly interlaced with our glycowork platform and can be easily tested at https://colab.research.google.com/github/BojarLab/CandyCrunch/blob/main/CandyCrunch.ipynb . We envision CandyCrunch to democratize structural glycomics and the elucidation of biological roles of glycans., (© 2024. The Author(s).)

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 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 μs 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 glycan's 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., Competing Interests: CI, LN, CF, AH, YD, JK, EF No competing interests declared, (© 2024, Ives, Nguyen et al.)

Fragment screening is used to identify binding sites and leads in drug discovery, but it is often unclear which binding sites are functionally important. Here, data from 37 experiments, and 1309 protein structures binding to 1601 ligands were analysed. A method to group ligands by binding sites is introduced and sites clustered according to profiles of relative solvent accessibility. This identified 293 unique ligand binding sites, grouped into four clusters (C1-4). C1 includes larger, buried, conserved, and population missense-depleted sites, enriched in known functional sites. C4 comprises smaller, accessible, divergent, missense-enriched sites, depleted in functional sites. A site in C1 is 28 times more likely to be functional than one in C4. Seventeen sites, which to the best of our knowledge are novel, in 13 proteins are identified as likely to be functionally important with examples from human tenascin and 5-aminolevulinate synthase highlighted. A multi-layer perceptron, and K-nearest neighbours model are presented to predict cluster labels for ligand binding sites with an accuracy of 96% and 100%, respectively, so allowing functional classification of sites for proteins not in this set. Our findings will be of interest to those studying protein-ligand interactions and developing new drugs or function modulators., (© 2024. The Author(s).)

The selective exchange of ions across cellular membranes is a vital biological process. Ca2+-mediated signaling is implicated in a broad array of physiological processes in cells, while elevated intracellular concentrations of Ca2+ are cytotoxic. Due to the significance of this cation, strict Ca2+ concentration gradients are maintained across the plasma and organelle membranes. Therefore, Ca2+ signaling relies on permeation through selective ion channels that control the flux of Ca2+ ions. A key family of Ca2+-permeable membrane channels is the polymodal signal-detecting transient receptor potential (TRP) ion channels. TRP channels are activated by a wide variety of cues including temperature, small molecules, transmembrane voltage, and mechanical stimuli. While most members of this family permeate a broad range of cations non-selectively, TRPV5 and TRPV6 are unique due to their strong Ca2+ selectivity. Here, we address the question of how some members of the TRPV subfamily show a high degree of Ca2+ selectivity while others conduct a wider spectrum of cations. We present results from all-atom molecular dynamics simulations of ion permeation through two Ca2+-selective and two non-selective TRPV channels. Using a new method to quantify permeation cooperativity based on mutual information, we show that Ca2+-selective TRPV channel permeation occurs by a three-binding site knock-on mechanism, whereas a two-binding site knock-on mechanism is observed in non-selective TRPV channels. Each of the ion binding sites involved displayed greater affinity for Ca2+ over Na+. As such, our results suggest that coupling to an extra binding site in the Ca2+-selective TRPV channels underpins their increased selectivity for Ca2+ over Na+ ions. Furthermore, analysis of all available TRPV channel structures shows that the selectivity filter entrance region is wider for the non-selective TRPV channels, slightly destabilizing ion binding at this site, which is likely to underlie mechanistic decoupling., (© 2023 Ives et al.)

Gram-negative bacteria cause the majority of highly drug-resistant bacterial infections. To cross the outer membrane of the complex Gram-negative cell envelope, antibiotics permeate through porins, trimeric channel proteins that enable the exchange of small polar molecules. Mutations in porins contribute to the development of drug-resistant phenotypes. In this work, we show that a single point mutation in the porin PorB from Neisseria meningitidis, the causative agent of bacterial meningitis, can strongly affect the binding and permeation of beta-lactam antibiotics. Using X-ray crystallography, high-resolution electrophysiology, atomistic biomolecular simulation, and liposome swelling experiments, we demonstrate differences in drug binding affinity, ion selectivity and drug permeability of PorB. Our work further reveals distinct interactions between the transversal electric field in the porin eyelet and the zwitterionic drugs, which manifest themselves under applied electric fields in electrophysiology and are altered by the mutation. These observations may apply more broadly to drug-porin interactions in other channels. Our results improve the molecular understanding of porin-based drug-resistance in Gram-negative bacteria., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The bifunctional undecaprenol kinase/phosphatase (UdpK) is a small, prokaryotic, integral membrane kinase, homologous with Escherichia coli diacylglycerol kinase and expressed by the dgkA gene. In Gram-positive bacteria, UdpK is involved in the homeostasis of the bacterial undecaprenoid pool, where it converts undecaprenol to undecaprenyl phosphate (C 55 P) and also catalyses the reverse process. C 55 P is the universal lipid carrier and critical to numerous glycopolymer and glycoprotein biosynthetic pathways in bacteria. DgkA gene expression has been linked to facilitating bacterial growth and survival in response to environmental stressors, as well being implicated as a resistance mechanism to the topical antibiotic bacitracin, by providing an additional route to C 55 P. Therefore, identification of UdpK inhibitors could lead to novel antibiotic treatments. A combination of homology modelling and mutagenesis experiments on UdpK have been used to identify residues that may be involved in kinase/phosphatase activity. In this review, we will summarise recent work on the mechanism and substrate specificity of UdpK., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The transport of charged molecules across biological membranes faces the dual problem of accommodating charges in a highly hydrophobic environment while maintaining selective substrate translocation. This has been the subject of a particular controversy for the exchange of ammonium across cellular membranes, an essential process in all domains of life. Ammonium transport is mediated by the ubiquitous Amt/Mep/Rh transporters that includes the human Rhesus factors. Here, using a combination of electrophysiology, yeast functional complementation and extended molecular dynamics simulations, we reveal a unique two-lane pathway for electrogenic NH 4 + transport in two archetypal members of the family, the transporters AmtB from Escherichia coli and Rh50 from Nitrosomonas europaea . The pathway underpins a mechanism by which charged H + and neutral NH 3 are carried separately across the membrane after NH 4 + deprotonation. This mechanism defines a new principle of achieving transport selectivity against competing ions in a biological transport process., Competing Interests: GW, GT, AB, MB, GD, MB, AP, CI, ET, PH, AM, UZ, AJ No competing interests declared, (© 2020, Williamson et al.)