Biology subjects

Holyoak, T.

Publications and source records attributed to Holyoak, T..

7 recordsLinked to original sources

bioRxiv2026

AI-based structural modelling and pathogenic variant mapping across the human protein interactome

Spanning the complex landscape of the human proteome, physical interactions between proteins coordinate cellular organization, signaling, regulation, and homeostasis. Yet the three-dimensional structures of most of these interactions remain unresolved. Here, we performed AI-based modelling of 109,282 human protein interaction structures, generating a structural resource for large-scale analysis. The predicted structures were systematically characterized through analysis of interface residue involvement, buried surface area, intermolecular bonding, and energetic properties. Additionally, we mapped pathogenic variant data onto the predicted structures, identifying interface-localized mutations for over 4,000 annotated disease categories. Acute myeloid leukemia, Alzheimer's disease, and cardiomyopathy were examined as representative disease contexts for exploring the distribution of interface mutations within disease-associated protein interaction networks. Strengths and limitations of predictive capabilities were also assessed, including through experimental structure comparisons. Collectively, this structural resource and accompanying analyses offer a basis for developing novel hypotheses and informing experimental studies involving human protein interactions in physiological and disease contexts.

bioinformatics↗

bioRxiv2026

Divergent Inclusion Body Structures and Stabilities Emerge from Native Monomer Properties

Protein aggregation is of broad importance in biotechnology and disease, yet the structural heterogeneity of cellular aggregates has confounded high-resolution structural analysis. Inclusion bodies (IBs) formed in Escherichia coli are an attractive, controllable system for unravelling the complexities of protein aggregation in a cellular context. Here, a multimodal analysis integrating residue-resolved quenched amide hydrogen-deuterium exchange (qHDX), proteolysis, FTIR, Congo red binding, and chemical denaturation is applied to IBs formed by proteins encompassing stable {beta}- and -globular folds, a partially structured protein fragment, and intrinsically disordered low complexity domains (LCDs). Remarkable conformational diversity is observed: IBs formed by well-folded proteins are extensively structured and include substantial local native-like features, whereas proteins with decreased access to stable native conformations form more heterogeneous and dynamic aggregates increasingly shaped by intrinsic sequence features. Strikingly, qHDX protection of TDP-43 LCD IBs strongly aligns with the core of cryo-EM structures of ex vivo pathological fibrils; however, peripheral regions that appear fully hydrogen-bonded in the cryo-EM structures exhibit little protection. The results reveal that individual protein IBs contain distinct mixtures of native-like, disordered, and amyloid-like conformers, informing the prediction and control of cellular aggregate structure and stability. Entry for the Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/729379v2_ufig1.gif" ALT="Figure 1"> View larger version (56K): org.highwire.dtl.DTLVardef@1542626org.highwire.dtl.DTLVardef@194ad5forg.highwire.dtl.DTLVardef@26c13org.highwire.dtl.DTLVardef@1055573_HPS_FORMAT_FIGEXP M_FIG Inclusion body conformational heterogeneity. Left: aggregates retaining local native-like structure. Middle: assemblies populated by -helical intermediates coexisting with antiparallel -sheet oligomers. Right: initially disordered states undergo helix-helix interactions and progress towards -sheet-rich conformations, illustrating distinct routes of cellular aggregation. C_FIG

biochemistry↗

bioRxiv2025

Structure of the Thomasclavelia ramosa immunoglobulin A protease reveals a modular and minimizable architecture distinct from other immunoglobulin A proteases

Immunoglobulin A proteases (IgAPs) are a diverse group of enzymes secreted from bacteria that inhabit human mucosal tissues. These enzymes have convergently evolved to cleave human immunoglobulin A as a means of modulating and evading host immunity. Only two of three known IgAP families have been biochemically characterized beyond their initial discovery. Here, we show using solution-scattering, steady-state kinetic, and crystallographic approaches that the protease from Thomasclavelia ramosa, representing the uncharacterized third family, has a truly modular and minimizable protein architecture. This analysis also revealed a unique metal-associated domain that likely functions as a molecular spacer and generated a working hypothesis detailing the structural mechanism behind the enzymes high substrate specificity. Our work provides the first in-depth biochemical account of this IgAP family, paving the way for advancing clinically relevant IgAP-related research and our understanding of IgAPs as a whole.

biochemistry↗

bioRxiv2024

A structural perspective on the temperature-dependent activity of enzymes

Enzymes are biomolecular catalysts whose activity varies with temperature. Unlike for small-molecule catalysts, the structural ensembles of enzymes can vary substantially with temperature, and it is in general unclear how this modulates the temperature dependence of activity. Here multi-temperature X-ray crystallography was used to record structural changes from -20{degrees}C to 40{degrees}C for a mesophilic enzyme in complex with inhibitors mimicking substrate-, intermediate-, and product-bound states, representative of major complexes underlying the kinetic constant kcat. Both inhibitors, substrates and catalytically relevant loop motifs increasingly populate catalytically competent conformations as temperature increases. These changes occur even in temperature ranges where kinetic measurements show roughly linear Arrhenius/Eyring behavior where parameters characterizing the system are assumed to be temperature independent. Simple analysis shows that linear Arrhenius/Eyring behavior can still be observed when the underlying activation energy / enthalpy values vary with temperature, e.g., due to structural changes, and that the underlying thermodynamic parameters can be far from values derived from Arrhenius/Eyring model fits. Our results indicate a critical role for temperature-dependent atomic-resolution structural data in interpreting temperature-dependent kinetic data from enzymatic systems. One-Sentence SummaryStructural data spanning a 60{degrees}C temperature range for enzyme complexes mimicking the substrate-, intermediate-, and product-bound states illuminate how small temperature-dependent structural changes may modulate activity and render parameters deduced from Arrhenius/Eyring plots unreliable.

biophysics↗

bioRxiv2022

The H163A Mutation Unravels an Oxidized Conformation of the SARS-CoV-2 Main Protease and Opens a New Avenue for Anti-Viral Therapeutic Design

The main protease of SARS-CoV-2 (Mpro) is an important target for developing COVID-19 therapeutics. Recent work has highlighted Mpros susceptibility to undergo redox-associated conformational changes in response to cellular and immune-system-induced oxidation. Despite structural evidence indicating large-scale rearrangements upon oxidation, the mechanisms of conformational change and its functional consequences are poorly understood. Here, we present the crystal structure of a new Mpro point mutant (H163A) that shows an oxidized conformation with the catalytic cysteine in a disulfide bond. We hypothesize that Mpro adopts this conformation under oxidative stress to protect against over-oxidation. Our metadynamics simulations illustrated a potential mechanism by which H163 modulates this transition and suggest that this equilibrium exists in the wild-type enzyme. We show that other point mutations can also significantly shift the equilibrium towards this state by altering conformational free energies. New therapeutic strategies against SARS-CoV-2 can be explored by understanding how H163 modulates this equilibrium.

biochemistry↗

bioRxiv2021

Millisecond Mix-and-Quench Crystallography (MMQX) Enables Time-Resolved Studies of PEPCK With Remote Data Collection

Time-resolved crystallography of biomolecules in action has advanced rapidly as methods for serial crystallography have improved, but the large number of crystals and complex experimental infrastructure required remain serious obstacles to widespread application. We have developed Millisecond Mix-and-Quench crystallography (MMQX), which yields millisecond time-resolved data using far fewer crystals and routine remote synchrotron data collection. To demonstrate the capabilities of MMQX, the conversion of oxaloacetic acid to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase is observed with a time resolution of 40 ms. MMQX, by lowering the entry barrier to time-resolved crystallography, should enable broad expansion in structural studies of protein dynamics.

biochemistry↗

bioRxiv2019

Discovery of a redox-thiol switch regulating cellular energy metabolism

Previously, we reported that increased synthesis of the gas hydrogen sulfide (H2S) during the Integrated Stress Response (ISR) induced proteome-wide cysteine-sulfhydration with the predominant modified pathway being enzymes of cellular energy metabolism (Gao, et al. 2015). Using pancreatic beta cells and quantitative proteomics in this study, we identified a Redox Thiol Switch from S-glutathionylation to S-sulfhydration and we named it, RTSGS. About half of the identified proteins are involved in energy metabolism, and one novel target was the mitochondrial phosphoenolpyruvate carboxykinase 2 (PCK2) whose catalytic Cys306was targeted by both modifications. The enzymatic activity of PCK2 was inhibited by S-glutathionylation, and this inhibition was largely reversed by S-sulfhydration. S-sulfhydration also reversed the S-glutathionylation-mediated inhibition of glucose flux, indicating a broad metabolic significance. We propose that a Redox Thiol Switch from S-glutathionylation to S-sulfhydration is a key mechanism to fine tune cellular energy metabolism in response to different levels of oxidative stress.

biochemistry↗