Biology subjects

Marques, S. M.

Publications and source records attributed to Marques, S. M..

6 recordsLinked to original sources

bioRxiv2026

ProtXAI: Explainable AI Reveals Structural Determinants of Protein Dynamics

Molecular dynamics simulations provide atomistic views of protein motions, but conventional analyses often struggle with extracting subtle mechanistic insights from complex trajectories. Here, we present an integrated framework, ProtXAI, combining molecular dynamics and explainable artificial intelligence (XAI), to identify residue-level determinants of conformational change across diverse protein systems. By leveraging inter-residue distance dynamics, deep learning, and sequential relevance propagation, the approach captures both local fluctuations and long-range communication pathways within protein structures. We applied this framework to three mechanistically distinct systems: apolipoprotein E4 (ApoE4), staphylokinase (SAK) variants, and an ancestral luciferase. Across these applications, our XAI-based approach recovered experimentally supported dynamic hotspots: ligand-responsive hinges in ApoE4, mutation-dependent flexibility shifts in SAK, and evolutionary redistribution of motions in the luciferase. ProtXAI also revealed additional long-range couplings not accessible to classical analysis. Together, these findings demonstrate that combining molecular dynamics with XAI provides a general and scalable strategy for dissecting protein dynamics and uncovering structural determinants of function, stability, and evolutionary changes without prior bias. This approach thus advances the current methodological repertoire for analysing proteins and their intrinsic properties. HighlightsMolecular dynamics simulations are increasingly accessible, yet scalable tools for comparative analysis remain limited. We demonstrate that machine learning coupled with explainable AI can automatically extract structural determinants of protein dynamics from trajectories. ProtXAI identifies key dynamic regions across diverse scenarios, including comparison of protein variants, understanding ligand modulation, and single-trajectory analysis. ProtXAI enables scalable, unbiased interpretation of long trajectories, providing an alternative to manual, time-intensive analysis.

bioinformatics↗

bioRxiv2025

Molecular trick to reverse SN2 mechanism in hydrolytic enzyme

Hydrolytic haloalkane dehalogenase enzymes catalyze an SN2 nucleophilic substitution to erase halogen substituents in organohalogen compounds. The acid-base-nucleophile triad secures irreversible SN2 displacement of the halogen for the hydroxyl derived from the water. Catalysis relies on the protonatable imidazole ring of the histidine base, and its substitution with an asparagine traps the enzyme in a covalently bound intermediate state, a principle exploited in the widely used HaloTag technology. In contrast, the histidine-to-phenylalanine substitution triggers reversibility of the SN2 mechanism, but the molecular trick by which it reprograms the catalytic pathway remains unknown. Here, we show that the phenylalanine at the site of the histidine base spatially disturbs the adjacent residues, leading to the remodeling of surrounding active-site loops. Consequently, rerouting of the access tunnels imparts distinctive kinetic behavior, featuring a reversible SN2 chemical step that facilitates transhalogenation reactions. This information is crucial for engineering next-generation biocatalysts for sustainable chemistry. HighlightsO_LICatalytic triad (glutamate-histidine-aspartate) secures irreversible SN2 mechanism C_LIO_LIHistidine-to-phenylalanine substitution in the triad leads to a reversible SN2 reaction C_LIO_LIEnzyme active site rerouting is a hallmark of the catalytic reprogramming C_LIO_LIBasis for designing biocatalysts for sustainable transhalogenation chemistry C_LI The bigger pictureHaloalkane dehalogenases catalyze the hydrolytic cleavage of the carbon-halogen bond in halogenated hydrocarbons, a feature used in a wide variety of industrial and biotechnological processes. In HaloTag technology, the catalytic histidine is replaced by an asparagine to enable a stable covalent bonding between a probe and a protein for biological imaging, affinity purification, etc. Remarkably, this originally irreversible SN2 process becomes fully reversible in a histidine-to-phenylalanine mutant. Moreover, halogen ion product is released at this stage, offering a greener alternative for transhalogenation reactions compared to the conventional synthetic approach and a way to recycle environmentally harmful halogenated compounds. However, the optimization of enzymes is needed for applications on an industrial scale. Therefore, we investigated the kinetics of the SN2 step and structural features of these two enzyme mutants, providing a basis for subsequent optimization efforts.

bioengineering↗

bioRxiv2025

Taurine Inhibits Apolipoprotein E4 Aggregation

Apolipoprotein E4 (ApoE4) is a major genetic risk factor in many neurodegenerative diseases, yet effective therapeutic strategies targeting its associated pathologies remain unresolved. The aggregation of ApoE4, a key pathological feature, can be attenuated by tramiprosate and its metabolite 3-sulfopropanoic acid. In this study, we investigated the potential of taurine, a close chemical analogue of tramiprosate, to modulate ApoE4-mediated pathological processes. Using an integrated approach--including molecular dynamics simulations, static light scattering, mass spectrometry, and cerebral organoid models--we investigated taurines effects on ApoE4 aggregation. We found that taurine effectively inhibits ApoE4 aggregation. Notably, taurine significantly ameliorates the pathophysiological characteristics of ApoE4, bringing its phenotype closer to the more benign ApoE3 variant. By leveraging its neuroprotective properties, taurine may offer effects comparable to tramiprosate and 3-sulfopropanoic acid, positioning it as an accessible and promising candidate for mitigating neurodegeneration, particularly in individuals with the high-risk ApoE4/E4 genotype.

biochemistry↗

bioRxiv2023

Effects of Alzheimer's Disease Drug Candidates on Disordered Aβ42 Dissected by Comparative Markov State Analysis (CoVAMPnet)

Computational study of the effect of drug candidates on intrinsically disordered biomolecules is challenging due to their vast and complex conformational space. Here we developed a Comparative Markov State Analysis (CoVAMPnet) framework to quantify changes in the conformational distribution and dynamics of a disordered biomolecule in the presence and absence of small organic drug candidate molecules. First, molecular dynamics trajectories are generated using enhanced sampling, in the presence and absence of small molecule drug candidates, and ensembles of soft Markov state models (MSMs) are learned for each system using unsupervised machine learning. Second, these ensembles of learned MSMs are aligned across different systems based on a solution to an optimal transport problem. Third, the directional importance of inter-residue distances for the assignment to different conformational states is assessed by a discriminative analysis of aggregated neural network gradients. This final step provides interpretability and biophysical context to the learned MSMs. We applied this novel computational framework to assess the effects of ongoing phase 3 therapeutics tramiprosate (TMP) and its metabolite 3-sulfopropanoic acid (SPA) on the disordered A{beta}42 peptide involved in Alzheimers disease. Based on adaptive sampling molecular dynamics and CoVAMPnet analysis, we observed that both TMP and SPA preserved more structured conformations of A{beta}42 by interacting non-specifically with charged residues. SPA impacted A{beta}42 more than TMP, protecting -helices and suppressing the formation of aggregation-prone {beta}-strands. Experimental biophysical analyses showed only mild effects of TMP/SPA on A{beta}42, and activity enhancement by the endogenous metabolization of TMP into SPA. Our data suggest that TMP/SPA may also target other biomolecules than A{beta} peptides. The CoVAMPnet method is broadly applicable to study the effects of drug candidates on the conformational behavior of intrinsically disordered biomolecules. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/523007v2_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@13eea16org.highwire.dtl.DTLVardef@17a6bd1org.highwire.dtl.DTLVardef@3c6b33org.highwire.dtl.DTLVardef@a20444_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2022

Illuminating the mechanism and allosteric behavior of NanoLuc luciferase

NanoLuc, a superior {beta}-barrel fold luciferase, was engineered 10 years ago but the nature of its catalysis remains puzzling. Here experimental and computational techniques were combined, revealing that imidazopyrazinone luciferins bind to an intra-barrel catalytic site but also to an allosteric site shaped on the enzyme surface. Structurally, binding to the allosteric site prevents simultaneous binding to the catalytic site, and vice versa, through concerted conformational changes. We demonstrate that restructuration of the allosteric site can boost the luminescent reaction in the remote active site. Mechanistically, an intra-barrel arginine coordinates the imidazopyrazinone component of luciferin, which reacts with O2 via a radical charge-transfer mechanism, and then it also protonates the resulting excited amide product to form a light-emitting neutral species. Concomitantly, an aspartate, supported by two tyrosines, fine-tunes the blue color emitter to secure a high emission intensity. This information is critical to engineering the next-generation of ultrasensitive bioluminescent reporters.

biochemistry↗

bioRxiv2022

Domino-like Effect of C112R Mutation on APOE4 Aggregation and Its Suppression by Alzheimer's Disease Drug Candidate

BackgroundApolipoprotein E (ApoE) {varepsilon}4 genotype is the most prevalent risk factor for late-onset Alzheimers Disease (AD). Although ApoE4 differs from its non-pathological ApoE3 isoform only by the C112R mutation, the molecular mechanism of its proteinopathy is unknown. MethodsHere, we reveal the molecular mechanism of ApoE4 aggregation using a combination of experimental and computational techniques, including X-ray crystallography, site-directed mutagenesis, hydrogen-deuterium mass spectrometry (HDX-MS), static light scattering and molecular dynamics simulations. Treatment of ApoE {varepsilon}3/{varepsilon}3 and {varepsilon}4/{varepsilon}4 cerebral organoids with tramiprosate was used to compare the effect of tramiprosate on ApoE4 aggregation at the cellular level. ResultsWe found that C112R substitution in ApoE4 induces long-distance (>15 [A]) conformational changes leading to the formation of a V-shaped dimeric unit that is geometrically different and more aggregation-prone than the ApoE3 structure. AD drug candidate tramiprosate and its metabolite 3-sulfopropanoic acid induce ApoE3-like conformational behavior in ApoE4 and reduce its aggregation propensity. Analysis of ApoE {varepsilon}4/{varepsilon}4 cerebral organoids treated with tramiprosate revealed its effect on cholesteryl esters, the storage products of excess cholesterol. ConclusionsOur results connect the ApoE4 structure with its aggregation propensity, providing a new druggable target for neurodegeneration and ageing.

neuroscience↗