Biology subjects

Barducci, A.

Publications and source records attributed to Barducci, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Interplay Between Protein-RNA Binding and Phase Separation Drives Emergent Behavior in RNP Condensates

Protein-RNA binding and biomolecular condensation are two key processes underlying the assembly and function of ribonucleoprotein (RNP) condensates. However, the understanding of the physical consequences of their interplay is still incomplete. To investigate this coupling, here we develop a minimal coarse-grained molecular model that combines specific, saturable protein-RNA binding with multivalent protein-protein interactions. Our results show that RNA acts as a molecular scaffold whose ability to promote condensation depends on the distribution of bound proteins across RNA molecules. This provides a simple microscopic explanation for both RNA-length-dependent condensation and re-entrant phase behavior, showing that condensate dissolution at high RNA concentration can emerge from entropic effects without requiring explicit electrostatic interactions. Conversely, condensate assembly markedly enhances effective protein-RNA binding, demonstrating that substantial changes in binding behavior can emerge without changes in intrinsic affinity. This provides a general physical mechanism through which condensates can reshape molecular competition between RNA-binding proteins. Together, these findings establish a framework linking RNA binding and biomolecular condensation, illustrating how their interplay governs condensate assembly.

biophysics↗

bioRxiv2025

Evidence for the Progressive Improvement of All-Atom Force Fields in Reproducing Local Conformational Preferences of Flexible Peptides

AbstractClassical protein force fields are widely used to probe the conformational properties of intrinsically disordered regions, yet their accuracy in reproducing local structural preferences remains uneven. We evaluated seven Amber and CHARMM force fields across three generations using molecular dynamics simulations of glycine-X-glycine tripeptides, with guest residues that span diverse physicochemical properties. Conformational ensembles were compared against distributions of conformations extracted from the crystallographic structures in the Protein Data Bank, and a statistical model derived from NMR observables. Analysis of secondary structure populations and Ramachandran distributions analyzed via Wasserstein distances reveals a clear historical progression. Early models display strong helical bias, intermediate ones approach Protein Data Bank trends, and recent versions shift toward solution-like ensembles dominated by polyproline II structure. None of the force fields fully captures the experimental distributions, although recent models show marked improvement over earlier generations. The remaining discrepancies point to specific aspects of local structure that still require tuning, while the overall progress underscores a steady trajectory toward more reliable descriptions of disordered peptides.

biophysics↗

bioRxiv2025

The Heterogeneous Solution Ensemble of the DEAD-box Protein Dhh1 Reveals a Modular Architecture

DEAD-box proteins such as Dhh1 play essential roles in RNA metabolism and the formation of biomolecular condensates, with a modular architecture comprising folded domains and disordered regions. To elucidate how this architecture shapes conformational dynamics in solution, we combined solution scattering experiments and multi-scale simulations on core and full-length constructs. Enhanced-sampling simulations captured a dynamic ensemble of core conformations stabilized by transient interdomain contacts that underpin functional regulation. Coarse-grained modelling revealed that disordered tails behave as independent modules exerting minimal influence on core dynamics. This integrated approach reveals a modular organization balancing structural heterogeneity and functional specificity, providing a framework for studying DEAD-box proteins in phase separation.

biophysics↗

bioRxiv2025

Uncovering the thermodynamic principles of enzymaticregulation in biomolecular condensates with reactivesimulations

Biomolecular condensates are dynamic cellular assemblies stabilized by weak intermolecular interactions. Cells regulate condensate formation, composition, and function through energy-consuming processes such as post-translational modifications (PTMs), which modify the physicochemical properties of condensate components, thereby dynamically reshaping these interactions. Here, we investigate how enzymatic reactions regulate phase-separated systems using a thermodynamically consistent particle-based model, which allows sampling of out-of-equilibrium steady states. We find that reaction kinetics are intrinsically coupled to the local molecular environment, leading to the formulation of two general principles. First, reactions that weaken favorable interactions are thermodynamically suppressed within condensates. As a consequence, regulation of condensate solubility is most efficient when PTMs tune interactions to values close to the solubility threshold. In contrast, reaction rates are generally enhanced at condensate interfaces, where the thermodynamic inhibition is relieved, but reactant availability remains high. Higher-resolution simulations of FUS and DDX4 proteins indicate that this interfacial contribution remains substantial even for micron-sized condensates, positioning interfaces as key determinants of biochemical activity in phase-separated systems alongside the properties of the condensate bulk. Together, these findings identify general, thermodynamic principles that govern the regulation of biomolecular condensates and link enzymatic activity with phase separation. Moreover, they provide a thermodynamically consistent molecular framework that can be applied to a broad range of regulatory processes in active phase-separated systems.

biophysics↗

bioRxiv2024

Selective ion binding and uptake shape the microenvironment of biomolecular condensates

Biomolecular condensates modulate various ion-dependent cellular processes and can regulate subcellular ion distributions by selective uptake of ions. However, the molecular grammar governing condensate-ion interactions is poorly understood. Here, we use NMR spectroscopy of ions and model condensate components to quantify and spatially resolve selective ion binding to condensates and show that these interactions follow the law of matching water affinities, resulting in strong binding between proteins and chaotropic anions, and between nucleic acids and kosmotropic cations. Ion uptake into condensates directly follows binding affinities, resulting in selective uptake of strong-binding ions, but exclusion of weak-binding ions. Ion binding further shapes the condensate microenvironment by altering the composition, viscosity and interface potential. Such changes can have profound effects on biochemical processes taking place inside condensates, as we show for RNA duplex formation. Our findings provide a new perspective on the role of condensate-ion interactions in cellular bio- and electrochemistry and may aid design of condensate-targeting therapeutics.

biophysics↗

bioRxiv2024

Polysomes and mRNA control the biophysical properties of the eukaryotic cytoplasm

The organization and biophysical properties of the cytoplasm influence all cellular reactions, including molecular interactions and the mobility of biomolecules. It has become clear that the cytoplasm does not behave like a simple fluid but instead is a densely crowded and highly organized environment. Yet, the detailed properties of the cytoplasm, the molecular mechanisms that control them and how they influence the biochemistry of cells remain poorly understood. Here, we investigate the diffusive properties of the cytoplasm in silico and in vivo, employing mRNPs (messenger ribonucleoprotein) and GEM (genetically encoded multimeric) particles as rheological probes in proliferating cells. We demonstrate that cytoplasmic diffusivity increases upon polysome disassembly due to translation inhibition or upon a reduction in mRNA levels. Reducing ribosome concentration by up to 20-25% without a change in polysome levels has no effect in vivo. In addition, we show that upon polysome disassembly, mRNA condensation into P-bodies does not affect cytosolic diffusion in budding yeast. Altogether, our results show that mRNAs and their organization into polysomes control the biophysical properties of the eukaryotic cytoplasm. HighlightsO_LIPolysomes control the biophysical properties of cytoplasm. C_LIO_LImRNP and GEM mobility is enhanced upon translation inhibition that leads to polysome disassembly C_LIO_LIPerturbation of mRNA levels leads to an increase in cytosolic diffusion. C_LI

cell biology↗

bioRxiv2024

Exploring RNA destabilization mechanisms in biomolecular condensates through atomistic Simulations

Biomolecular condensates are currently recognized to play a key role in organizing cellular space and in orchestrating biochemical processes. Despite an increasing interest in characterizing their internal organization at the molecular scale, not much is known about how the densely crowded environment within these condensates affects the structural properties of recruited macromolecules. Here we adopted explicit-solvent all-atom simulations based on a combination of enhanced sampling approaches to investigate how the conformational ensemble of an RNA hairpin is reshaped in a highlyconcentrated peptide solution that mimics the interior of a biomolecular condensates. Our simulations indicate that RNA structure is greatly perturbed by this distinctive physico-chemical environment, which weakens RNA secondary structure and promotes extended non-native conformations. The resulting high-resolution picture reveals that RNA unfolding is driven by the effective solvation of nucleobases through hydrogen bonding and stacking interactions with surrounding peptides. This solvent effect can be modulated by the aminoacid composition of the model condensate as proven by the differential RNA behaviour observed in the case of arginine-rich and lysine-rich peptides.

biophysics↗

bioRxiv2022

Nucleation of Biomolecular Condensates from Finite-Sized Simulations

The nucleation of protein condensates is a concentration-driven process of assembly. When modelled in the canonical ensemble, condensation is affected by finite-size effects. Here, we present a general and efficient route to obtain ensemble properties of protein condensates in the macroscopic limit from finite-sized nucleation simulations. The approach is based on a theoretical description of droplet nucleation in the canonical ensemble and enables estimating thermodynamic and kinetic parameters, such as the macroscopic equilibrium density of the dilute protein phase, the condensates surface tension and nucleation free energy barriers. We apply the method to coarse-grained simulations of NDDX4 and FUS-LC, two phase-separating disordered proteins with different physicochemical characteristics. Our results show that NDDX4 condensate droplets, characterised by lower surface tension, higher solubility, and faster monomer exchange dynamics than FUS-LC, form with negligible nucleation barriers. In contrast, FUS-LC condensates form via an activated process over a wide range of concentrations. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=196 SRC="FIGDIR/small/515961v2_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@15306c9org.highwire.dtl.DTLVardef@1e2927forg.highwire.dtl.DTLVardef@1ffe65borg.highwire.dtl.DTLVardef@16c74e1_HPS_FORMAT_FIGEXP M_FIG Graphical Table of Contents. C_FIG

biophysics↗

bioRxiv2022

The structure of pathogenic huntingtin exon-1 defines the bases of its aggregation propensity

Huntingtons Disease is a neurodegenerative disorder caused by a CAG expansion of the first exon of the HTT gene, resulting in an extended poly-glutamine (poly-Q) tract in the N-terminus of the protein huntingtin (httex1). The structural changes occurring to the poly-Q when increasing its length remain poorly understood mainly due to its intrinsic flexibility and the strong compositional bias of the protein. The systematic application of site-specific isotopic labeling has enabled residue-specific NMR investigations of the poly-Q tract of pathogenic httex1 variants with 46 and 66 consecutive glutamines. The integrative analysis of the data reveals that the poly-Q tract adopts long -helical conformations stabilized by glutamine side-chain to backbone hydrogen bonds. 19F-NMR of site-specifically incorporated fluoro-glutamines and molecular dynamics simulations demonstrate that the mechanism propagating -helical conformations towards the poly-Q from the upstream N17 domain is independent of the poly-Q track length. Aggregation and atomic force microscopy experiments show that the presence of long and persistent -helices in the poly-Q tract is a stronger signature in defining the aggregation kinetics and the structure of the resulting fibrils than the number of glutamines. The ensemble of our observations provides a structural perspective of the pathogenicity of expanded httex1 and paves the way to a deeper understanding of poly-Q related diseases.

biophysics↗

bioRxiv2022

SLX4 assembles nuclear condensates that compartmentalize the SUMO-RNF4 pathway and drive DNA repair.

SLX4, disabled in Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in replication-coupled repair of DNA interstrand crosslinks (ICLs). Here we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.

biochemistry↗

bioRxiv2022

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR complex

SummaryLiving organisms have developed protein sensors helping them to adapt to their environment1. The aryl hydrocarbon receptor (AHR) is an emblematic member of this class of proteins, and a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites2. However, in the absence of high-resolution structural data, a molecular understanding of how AHR is activated by such diverse compounds is lacking. Here we present a 2.85 [A] cryo-electron microscopy structure of the cytosolic complex comprising AHR bound to the ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2. The structure reveals a closed Hsp90 dimer with AHR threaded through its lumen. XAP2 directly interacts with Hsp90 and the AHR ligand-binding domain, thereby acting as a brace stabilizing the entire complex. Importantly, we provide the first experimental visualization of the AHR PAS-B domain bound to a ligand, revealing a unique organization of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing unprecedented structural details of the molecular initiating event leading to AHR activation, our study rationalizes prior biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

biochemistry↗

bioRxiv2022

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins

The 70 kDalton Heat shock protein (Hsp70) chaperone system is emerging as a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform a vast array of different cellular functions is regulated by a family of co-chaperones known as J-domain proteins (JDP) that bear a small namesake J-domain, which is required to interact and drive the ATPase cycle of Hsp70s. Both prokaryotic and eukaryotic JDPs display staggering diversity in domain architecture (besides the ubiquitous J-domain), function, and cell localization. On the contrary, a relatively small number of Hsp70 paralogs exist in cells, suggesting a high degree of specificity, but also promiscuity, in the partnering between JDPs and Hsp70s. Very little is known about the JDP family, despite their essential role in cellular proteostasis, development, and the link to a broad range of human diseases. The number of JDP gene sequences identified across all kingdoms as a consequence of advancements in sequencing technology has exponentially increased, where it is now beyond the ability of careful manual curation. In this work, we first provide a broad overview of the JDP repertoire accessible from public databases, and then we use an automated classification scheme, based on Artificial Neural Networks (ANNs), to demonstrate that the sequences of J-domains carry sufficient discriminatory information to recover with high reliability the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match to residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

bioinformatics↗

bioRxiv2021

Functional control of a 0.5 MDa TET aminopeptidase by a flexible loop revealed by MAS NMR

Large oligomeric enzymes control a myriad of cellular processes, from protein synthesis and degradation to metabolism. The 0.5 MDa large TET2 aminopeptidase, a prototypical protease important for cellular homeostasis, degrades peptides within a ca. 60 [A] wide tetrahedral chamber with four lateral openings. The mechanisms of substrate trafficking and processing remain debated. Here, we integrate magic-angle spinning (MAS) NMR, mutagenesis, co-evolution analysis and molecular dynamics simulations and reveal that a loop in the catalytic chamber is a key element for enzymatic function. The loop is able to stabilize ligands in the active site and may additionally have a direct role in activating the catalytic water molecule whereby a conserved histidine plays a key role. Our data provide a strong case for the functional importance of highly dynamic - and often overlooked - parts of an enzyme, and the potential of MAS NMR to investigate their dynamics at atomic resolution.

biophysics↗

bioRxiv2021

Arginine multivalency stabilizes protein/RNA condensates

Biomolecular condensates assembled through liquid-liquid phase separation (LLPS) of proteins and RNAs are currently recognized to play an important role in cellular organization. Their assembly depends on the formation of a network of transient, multivalent interactions between flexible scaffold biomolecules. Understanding how protein and RNA sequences determine these interactions and ultimately regulate the phase separation is an open key challenge. Recent in vitro studies have revealed that arginine and lysine residues, which are enriched in most cellular condensates, have markedly distinct propensities to drive the LLPS of protein/RNA mixtures. Here, we employ explicit-solvent atomistic Molecular Dynamics (MD) simulations to shed light on the microscopic origin of this difference by investigating mixtures of polyU oligonucleotides with either polyR/polyK peptides. In agreement with experiments, our simulations indicate that arginine has a higher affinity for polyU than lysine both in highly diluted conditions and in concentrated solutions with a biomolecular density comparable to cellular condensate. The analysis of intermolecular contacts suggests that this differential behavior is due to the propensity of arginine side chains to simultaneously form a higher number of specific interactions with oligonucleotides, including hydrogen bonds and stacking interactions. Our results provide a molecular description of how the multivalency of the guanidinium group enables the coordination of multiple RNA groups by a single arginine residue, thus ultimately stabilizing protein/RNA condensates.

biophysics↗

bioRxiv2020

Unraveling molecular interactions in a phase-separating protein by atomistic simulations

Membraneless organelles are dynamical cellular condensates formed by the liquid-liquid phase separation of proteins and RNA molecules. Multiple evidence suggests that disordered proteins are structural scaffolds that drive the condensation by forming a dynamic network of inter- and intra-molecular contacts. Despite the blooming research activity in this field, the structural characterization of these condensates is very limited and we still do not understand how the phase behaviour is encoded in the amino-acid sequences of the scaffolding proteins. Here we exploited explicit-solvent atomistic simulations to disentangle the molecular interactions governing the phase behaviour of the N-terminal disordered region of DEAD-box helicase 4 (NDDX4), which is a well-established model for phase separation in vitro and in vivo. Single-molecule simulations clarified the interplay between the intramolecular interactions that shape NDDX4 conformational ensemble and the known determinants of its phase behaviour, such as the attraction between oppositely-charged regions and the presence of arginine and phenylalanine. We then investigated intermolecular interactions associated with phase separation via a divide-and-conquer strategy based on the simulations of various NDDX4 fragments at high concentration. Our approach allowed us to probe conditions mimicking real condensates and revealed, in agreement with mutagenesis results, how these interactions arise from the complex interplay of diverse molecular mechanisms. Particularly, we characterized the transient formation of clusters of arginine and aromatic residues, which may stabilize the assembly of several MLOs. Overall, our results reveal the potential of atomistic simulations in the investigation of biomolecular phase separation paving the way for future studies.

biophysics↗

bioRxiv2018

Interplay of protein disorder in retinoic acid receptor heterodimer and its corepressor regulates gene expression

The retinoic acid receptors (RARs) form heterodimers with retinoid X receptors (RXRs) and control gene transcription in response to ligand binding and via allosteric activation of the C-termini helix (helix H12) of its ligand-binding domain. Herein we show that in the absence of ligand, helices H12 of RXR and RAR are disordered. The selective RAR agonist, Am580, induces folding of H12, whereas in the presence of the inverse agonist BMS493, H12 stays mostly disordered. These results substantiate a link between the structural dynamics of H12 and RXR/RAR heterodimer biological functions, and highlight disordered-to-order transition as an essential mechanism for retinoic acid mediated regulation. Unliganded RAR exerts a strong repressive activity allowed by the recruitment of transcriptional corepressors and establishment of a corepressor complex in the promoter region of target genes. The human regulatory complex of the RAR bound to the full-length interaction domain of the corepressor N-CoR was studied by integrating several experimental (SAXS, X-ray crystallography, NMR, CD, AUC) and computational data. Unexpectedly, we found that, while mainly intrinsically disordered, the N-CoR presents partially evolutionary conserved structured regions that are involved in transient intramolecular contacts. In the presence of RXR/RAR, we show that N-CoR exploits its multivalency to form a multi-site complex that diplays an equilibrium between different conformational states. This conformational equilibrium is modulated by cognate ligands, RAR point mutation and RXR H12 deletion. Now, we can state that, in addition to NR conformation and ligand-induced allosteric changes, intrinsic disorder is substantially embedded in the synergetic regulation of RXR/RAR activity and its resulting abilities to communicate with the intracellular components.

biophysics↗

bioRxiv2018

DNA-segment-capture model for loop extrusion by structural maintenance of chromosome (SMC) protein complexes

Cells possess remarkable control of the folding and entanglement topology of long and flexible chromosomal DNA molecules. It is thought that structural maintenance of chromosome (SMC) protein complexes play a crucial role in this, by organizing long DNAs into series of loops. Recent experimental data suggest that SMC complexes are able to translocate on DNA, as well as pull out lengths of DNA via a \"loop extrusion\" process. We describe a Brownian loop-capture-ratchet model for translocation and loop extrusion based on known structural, catalytic, and DNA-binding properties of the Bacillus subtilis SMC complex. Our model provides an example of a new class of molecular motor where large conformational fluctuations of the motor track - in this case DNA - are involved in the basic translocation process. Quantitative analysis of our model leads to a series of predictions for the motor properties of SMC complexes, most strikingly a strong dependence of SMC translocation velocity on tension in the DNA track that it is moving along, with \"stalling\" occuring at subpiconewton tensions. A closely related hallmark of the loop-capture mechanism is a strong dependence of translocation step size on DNA tension. We also discuss how the same mechanism might be used by other structurally related SMC complexes (E. coli MukBEF and eukaryote condensin, cohesin and SMC5/6) to organize genomic DNA.

biophysics↗

bioRxiv2017

A catch-bond drives stator mechanosensitivity in the Bacterial Flagellar Motor

The bacterial flagellar motor (BFM) is the rotary motor which powers the swimming and swarming of many motile bacteria. The torque is provided by stator units, ion motive force powered ion channels known to assemble and disassemble dynamically in the BFM. This turnover is mechano-sensitive, with the number of engaged units dependent upon the viscous load experienced by the motor through the flagellum. However, the molecular mechanism driving BFM mechano-sensitivity is unknown. Here we directly measure the kinetics of arrival and departure of the stator units in individual wild-type motors via analysis of high-resolution recordings of motor speed, while dynamically varying the load on the motor via external magnetic torque. Obtaining the real-time stator stoichiometry before and after periods of forced motor stall, we measure both the number of active stator units at steady-state as a function of the load and the kinetic association and dissociation rates, by fitting the data to a reversible random sequential adsorption model. Our measurements indicate that BFM mechano-sensing relies on the dissociation rate of the stator units, which decreases with increasing load, while their association rate remains constant. This implies that the lifetime of an active stator unit assembled within the BFM increases when a higher force is applied to its anchoring point in the cell wall, providing strong evidence that a catch-bond mechanism can explain the mechano-sensitivity of the BFM.

biophysics↗