Biology subjects

Pandini, A.

Publications and source records attributed to Pandini, A..

7 recordsLinked to original sources

bioRxiv2026

Spatiotemporal graph neural networks reveal conformational binding signature in protein dynamics

Biomolecular function is often controlled by structural and dynamical adaptations to binding events. Although molecular dynamics (MD) simulations can capture these events at atomic resolution, separating functional signatures from stochastic noise remains challenging. Traditional methods often struggle to isolate mechanistically relevant differences across independent replicas. Here, we introduce an explainable deep learning approach that learns state-specific dynamic signatures directly from MD trajectories. By coupling a dynamic protein graph representation with group-aware contrastive learning across independent replicas, the model detects the signatures, filtering out trajectory-specific correlations. An explainable AI framework then maps the identified differences on individual residues. We demonstrate this approach by identifying "binding-ready" conformations in a T4-Lysozyme mutant, recovering the allosteric determinants of peptide recognition in the PDZ3 domain, and isolating a ligand-independent activation signature for the A2A receptor. Our GISTnet-MD method generalizes across unseen data during comparative MD analysis, translating raw trajectory differences into residue-level determinants of protein function.

biophysics↗

bioRxiv2025

Binding Affinity Ranking at the Molecular Initiating Event (BARMIE): An open-source computational pipeline for ecological hazard ranking of endocrine disrupting chemicals.

A challenge in ecological risk assessment is identifying the chemicals that pose the greatest threat and determining which species are most vulnerable to them. To help address this, this study has developed an in-silico open-source tool called BARMIE (Binding Affinity Ranking at the Molecular Initiating Event) to rapidly predict the chemical binding affinity of steroid receptor proteins to synthetic steroids to identify potentially vulnerable species and chemicals of concern. BARMIE was used to screen 163 teleost fish glucocorticoid receptors (GRs) for binding to the natural ligand cortisol and to 10 synthetic glucocorticoid drugs (GCs) designed to interact within the ligand-binding pocket (LBP) of GRs. BARMIE identified species from the superorder Protacanthopterygii with high-affinity GRs to synthetic GCs (e.g. vulnerable species). . BARMIE was also used to screen binding profiles of compounds in the Medicine for Malaria Venture Global Health Priority Box to rainbow trout GRs (rtGR1 and rtGR2). Of the 178 compounds, 24 and 36 bind within the LBP of rtGR1 and rtGR2, respectively. For 30 of these compounds, transactivation activity was assessed at 1{micro}M in the presence or absence of 1{micro}M cortisol and confirmed 2 compounds with agonistic properties (e.g. chemicals of concern) that would require further in vitro and/or in vivo studies to assess the environmental risk. BARMIE can rapidly generate predicted binding affinities for 100s of species and chemicals as a first screen in environmental risk assessment to provide information on which substances to prioritise in downstream tests.

pharmacology and toxicology↗

bioRxiv2024

Functionally Important Residues from Graph Analysis of Coevolved Dynamic couplings

The relationship between protein dynamics and function is essential for understanding biological processes and developing effective therapeutics. Functional sites within proteins are critical for activities such as substrate binding, catalysis, and structural changes. Existing computational methods for the predictions of functional residues are trained on sequence, structural and experimental data, but they do not explicitly model the influence of evolution on protein dynamics. This overlooked contribution is essential as it is known that evolution can fine tune protein dynamics through compensatory mutations, either to improve the proteins performance or diversify its function while maintaining the same structural scaffold. To model this critical contribution, we introduce DyNoPy, a computational method that combines residue coevolution analysis with molecular dynamics (MD) simulations, revealing hidden correlations between functional sites. DyNoPy constructs a graph model of residue-residue interactions, identifies communities of key residue groups and annotates critical sites based on their roles. By leveraging the concept of coevolved dynamical couplings--residue pairs with critical dynamical interactions that have been preserved during evolution--DyNoPy offers a powerful method for predicting and analysing protein evolution and dynamics. We demonstrate the effectiveness of DyNoPy on SHV-1 and PDC-3, chromosomally encoded {beta}-lactamases linked to antibiotic resistance, highlighting its potential to inform drug design and address pressing healthcare challenges.

evolutionary biology↗

bioRxiv2021

N4BP1 is dimerization-dependent linear ubiquitin reader regulating TNFR1 signalling through linear ubiquitin binding and Caspase-8-mediated processing

Signalling through TNFR1 modulates proinflammatory gene transcription and programmed cell death, and its impairment causes autoimmune diseases and cancer. NEDD4-binding protein 1 (N4BP1) was recently identified as a critical suppressor of proinflammatory cytokine production1, whose mode of action remained unknown. Here, we show that N4BP1 is a novel linear ubiquitin reader that negatively regulates NF{kappa}B signalling by its unique dimerizationdependent ubiquitin-binding module that we named LUBIN. Dimeric N4BP1 strategically positions two non-selective ubiquitin-binding domains to ensure exclusive recognition of linear ubiquitin. Under proinflammatory conditions, N4BP1 is recruited to the nascent TNFR1 signalling complex, where it regulates duration of proinflammatory signalling in LUBIN-dependent manner. N4BP1 deficiency accelerates TNF-induced cell death by increasing complex II assembly. Under proapoptotic conditions, Caspase-8 mediates proteolytic processing of N4BP1 and the resulting cleavage fragment of N4BP1, which retains the ability to bind linear ubiquitin, is rapidly degraded by the 26S proteasome, accelerating apoptosis. In summary, our findings demonstrate that N4BP1 dimerization creates a unique linear ubiquitin reader that ensures timely and coordinated regulation of TNFR1-mediated inflammation and cell death.

biochemistry↗

bioRxiv2021

Structural dynamics of the β-coronavirus Mpro protease ligand binding sites

{beta}-coronaviruses alone have been responsible for three major global outbreaks in the 21st century. The current crisis has led to an urgent requirement to develop therapeutics. Even though a number of vaccines are available, alternative strategies targeting essential viral components are required as a back-up against the emergence of lethal viral variants. One such target is the main protease (Mpro) that plays an indispensible role in viral replication. The availability of over 270 Mpro X-ray structures in complex with inhibitors provides unique insights into ligand-protein interactions. Herein, we provide a comprehensive comparison of all non-redundant ligand-binding sites available for SARS-CoV2, SARS-CoV and MERS-CoV Mpro. Extensive adaptive sampling has been used to explore conformational dynamics employing convolutional variational auto encoder-based deep learning, and investigates structural conservation of the ligand binding sites using Markov state models across {beta}-coronavirus homologs. Our results indicate that not all ligand-binding sites are dynamically conserved despite high sequence and structural conservation across {beta}-coronavirus homologs. This highlights the complexity in targeting all three Mpro enzymes with a single pan inhibitor.

biophysics↗

bioRxiv2019

Allosteric priming of E. coli CheY by the flagellar motor protein FliM

Phosphorylation of Escherichia coli CheY protein transduces chemoreceptor stimulation to a highly cooperative flagellar motor response. CheY binds to the N-terminal peptide of the FliM motor protein (FliMN). Constitutively active D13K-Y106W CheY has been an important tool for motor physiology. The crystal structures of CheY and CheY.FliMN with and without D13K-Y106W have shown FliMN bound CheY contains features of both active and inactive states. We used molecular dynamics (MD) simulations to characterize the CheY conformational landscape accessed by FliMN and D13K-Y106W. Mutual information measures identified the central features of the long-range CheY allosteric network between D13K at the D57 phosphorylation site and Y/W106 at the FliMN interface; namely the closure of the 4-{beta}4 hinge and inward rotation of Y/W106 with W58. We used hydroxy-radical foot-printing with mass spectroscopy (XFMS) to track the solvent accessibility of these and other sidechains. The solution XFMS oxidation rate correlated with the solvent-accessible area of the crystal structures. The protection of allosteric relay sidechains reported by XFMS confirmed the intermediate conformation of the native CheY.FliMN complex, the inactive state of free D13K-Y106W CheY and the MD-based network architecture. We extended the MD analysis to determine temporal coupling and energetics during activation. Coupled aromatic residue rotation was a graded rather than a binary switch with Y/W106 sidechain burial correlated with increased FliMN affinity. Activation entrained CheY fold stabilization to FliMN affinity. The CheY network could be partitioned into four dynamically coordinated community sectors. Residue substitutions mapped to sectors around D57 or the FliMN interface according to phenotype. FliMN increased sector size and interactions. These sectors fused between the substituted K13K-W106 residues to organize a tightly packed core and novel surfaces that may bind additional sites to explain the cooperative motor response. The community maps provide a more complete description of CheY priming than proposed thus far. Statement of SignificanceCheY affinity for FliMN, its binding target at the flagellar motor, is increased by phosphorylation to switch rotation sense. Atomistic simulations based on CheY and CheY.FliMN crystal structures with and without the phospho-mimetic double substitution (D13K-Y106W) showed CheY compaction is entrained to increased FliMN affinity. Burial of exposed aromatic sidechains drove compaction, as validated by tracking sidechain solvent accessibility with hydroxyl-radical foot-printing. The substitutions were localized at the phosphorylation pocket (D13K) and FliMN interface (Y106W). Mutual information measures revealed these locations were allosterically coupled by a specialized conduit when the conformational landscape of FliMN-tethered CheY was modified by the substitutions. Novel surfaces stabilized by the conduit may bind additional motor sites, essential for the high cooperativity of the flagellar switch.

biophysics↗

bioRxiv2019

Conformational coupling by trans-phosphorylation in calcium calmodulin dependent kinase II

The calcium calmodulin dependent protein kinase II (CaMKII) is a dodecameric holoenzyme important for encoding memory. Its activation, triggered by binding of calcium calmodulin, persists autonomously after calmodulin dissociation. One (receiver) kinase captures and subsequently phosphorylates the regulatory domain peptide of a donor kinase forming a chained dimer as a first stage of autonomous activation. Protein dynamics simulations examined the conformational changes triggered by dimer formation and phosphorylation, aimed to provide a molecular rationale for human mutations that result in learning disabilities. Ensembles generated from X-ray crystal structures were characterized by network centrality and community analysis. Mutual information related collective motions to local fragment dynamics encoded with a structural alphabet. Implicit solvent tCONCOORD conformational ensembles revealed the dynamic architecture of Inactive kinase do-mains was co-opted in the activated dimer but the network hub shifted from the nucleotide binding cleft to the captured peptide. Explicit solvent molecular dynamics (MD) showed nucleotide and substrate binding determinants formed coupled nodes in long-range signal relays between regulatory peptides in the dimer. Strain in the extended captured peptide was balanced by reduced flexibility of the receiver kinase C-lobe core. The relays were organized around a hydrophobic patch between the captured peptide and a key binding helix. The human mutations aligned along the relays. Thus, these mutations could disrupt the allosteric network alternatively, or in addition, to altered binding affinities. Non-binding protein sectors distant from the binding sites mediated the allosteric signalling; providing possible targets for inhibitor design. Phosphorylation of the peptide modulated the dielectric of its binding pocket to strengthen the patch, non-binding sectors, domain interface and temporal correlations between parallel relays. These results provide the molecular details underlying the reported positive kinase cooperativity to enrich discussion on how autonomous activation by phosphorylation leads to long-term behavioural effects.

neuroscience↗