Biology subjects

Bernado, P.

Publications and source records attributed to Bernado, P..

15 recordsLinked to original sources

bioRxiv2026

Generative design of intrinsically disordered proteins based on conditioned protein language models: Data is the limit

Intrinsically disordered proteins and regions (IDRs) are central to a multitude of biological processes. Despite extensive studies of their structural and physicochemical properties, the rational design of IDRs with defined conformational behavior remains challenging due to their ensemble nature. Here we present a generative framework for designing disordered protein sequences conditioned on target conformational ensemble descriptors using protein language models (pLMs). We formulate IDR design as the task of generating amino acid sequences predicted to realize specified biophysical properties and implement a Transformer encoder-decoder architecture that maps numerical descriptors to protein sequences. By training models on datasets spanning two orders of magnitude in size, we show that accurate control of conformational and physicochemical properties is achieved only at large data scale. These results demonstrate the feasibility of conditioning generative models on ensemble-level descriptors for IDR design. More broadly, these results support a data-centric paradigm for protein engineering, in which data availability emerges as a key limiting factor for the accurate design of IDRs.

bioinformatics↗

bioRxiv2026

Conformational ensembles of flexible multidomain proteins: How close are we to accurate and reliable predictions?

Multidomain proteins connected by flexible linkers populate conformational ensembles that are challenging to characterize using conventional structural biology methods. In domain-linker-domain (DLD) proteins, linker-mediated inter-domain relative positions and orientations are functionally relevant, yet their dynamical behavior in solution normally remain poorly described. Small-angle X-ray scattering (SAXS) provides ensemble-averaged structural information for such systems; however, coupling with computational modeling is required to accurately describe the dynamic behavior of this family of proteins in solution. Here, we present a systematic evaluation of five ensemble-generation strategies applied to a set of eighteen proteins sharing the same two globular domains, connected by naturally occurring linkers of varying length and composition. Modeling methods based on different underlying principles are compared by assessing their agreement to experimental SAXS data, showing a large disparity and systematic structural biases among them. Furthermore, for each approach, we examine the effect of refinement against SAXS restraints and assess its capacity to describe the experimental data, as well as the induced biases in global dimensions and inter-domain distance distributions. This analysis underlines the importance of the initial conformational pool for deriving experimentally compatible ensembles. Overall, this work provides a high-quality benchmark for SAXS-driven ensemble modeling of flexible, multidomain proteins and establishes a framework for the critical interpretation of solution scattering data in systems with pronounced conformational heterogeneity.

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

Interplay Between Intrinsically Disordered Proteins and Atomically Precise Gold Nanoclusters Modulates their Optical Properties

Understanding how structural and optical properties of metallic nanoclusters can be tuned by proteins is crucial for the use of these hybrid molecules in biomedical applications. The interaction of proteins with ultrasmall, atomically-precise gold nanoclusters (Au-NCs) has been mainly investigated in the context of structured proteins, while their behavior with intrinsically disordered proteins (IDPs) remains unexplored. This work examines the structural and optical properties of Au-NCs interacting with bioengineered IDPs containing up to three cysteines. We show that, by exploiting the conformational flexibility of cysteine-containing IDPs, we can anchor proteins to Au-NCs in a position-specific manner, leading to new bioconjugates with properties that differ from those of the individual components. We observed an up to 15-fold photoluminescence enhancement depending on the number of cysteines anchored. By combining mass spectrometry, small-angle X-ray scattering (SAXS), and computational modelling, the ensemble structures of nine bioconjugates with different stoichiometries were elucidated, indicating their overall compactness. Our results suggest that the interface between these atomically-precise species and the conformationally fluctuating protein is responsible for the optical properties of these nanobioconjugates. This research improves our understanding of Au-NC- protein interactions, paving the way to novel nano-molecular hybrid conjugates with tunable properties for bioimaging and therapeutic applications.

biochemistry↗

bioRxiv2025

New structural insights into the control of the retinoic acid receptors RAR/RXR by DNA, ligands and transcriptional coregulators

Retinoic acid receptors (RARs) are ligand-dependent transcription factors essential for various biological processes, including embryogenesis, differentiation, and apoptosis. RARs function as heterodimers with retinoid X receptors (RXRs) and regulate gene expression via retinoic acid response elements (RAREs). Their transcriptional activity is modulated by coregulators, with corepressors maintaining repression in the absence of ligand and coactivators enabling transcription upon ligand binding. Structural studies reveal that DNA binding induces conformational changes affecting coregulator interactions. However, the precise structural organization of RAR/RXR-coregulator complexes and the allosteric influence of DNA on receptor function remain incompletely understood. Our study presents an integrative analysis of the RAR/RXR heterodimer bound to four distinct and relevant RAREs (DR0, DR1, DR5, and IR0) in complex with either a corepressor (NCoR) or a coactivator (TIF-2) nuclear receptor interaction domain. By combining small-angle X-ray scattering, hydrogen/deuterium exchange mass spectrometry and molecular dynamics simulations, we revealed that the heterodimer adopts distinct conformations depending on the DNA sequence, influencing interdomain distances and receptor interactions. Additionally, we uncovered the dynamic interplay between ligand, DNA, and coregulator binding. This study provides new insights into the structural features of coregulator proteins and highlights the allosteric influence of RAREs on receptor function.

biochemistry↗

bioRxiv2023

Site-Specific Introduction of Alanines for the NMR Investigation of Low-Complexity Regions and Large Biomolecular Assemblies

NMR studies of large biomolecular machines and highly repetitive proteins remain challenging due to the difficulty of assigning signals to individual nuclei. Here, we present an efficient strategy to address this challenge by engineering a Pyrococcus horikoshii tRNA/alanyl-tRNA synthetase pair that enables the incorporation of up to three isotopically labeled alanine residues in a site-specific manner using in vitro protein expression. We have demonstrated the general applicability of this approach for NMR assignment by introducing isotopically labeled alanines into four proteins, including the 300-kDa molecular chaperone ClpP and the alanine-rich Phox2B transcription factor. For large protein assemblies, our labeling approach enables unambiguous assignments, while avoiding potential artefacts induced by site-specific mutations. When applied to Phox2B, which contains two poly-alanine tracts of nine and twenty alanines, we observe that the helical stability is strongly dependent on the homorepeat length, demonstrating structural cooperativity. The capacity to selectively introduce alanines with distinct labeling patterns is a powerful tool to probe structure and dynamics of biomolecular systems that are out of the reach of traditional structural biology methods.

biochemistry↗

bioRxiv2023

Phosphorylation motif dictates GPCR C-terminal domain conformation and arrestin interaction

Arrestin dependent G protein-coupled receptor (GPCR) signaling pathway is regulated by the phosphorylation state of GPCRs C-terminal domain, but the molecular bases of arrestin:receptor interaction are to be further illuminated. Here we investigated the impact of phosphorylation on the conformational features of the C-terminal region from three Rhodopsin-like GPCRs, the vasopressin V2 Receptor (V2R), the Growth Hormone Secretagogue or ghrelin Receptor type 1a (GHSR) and the {beta}2-Adernergic Receptor ({beta}2AR). Using phosphomimetic variants, we identified pre-formed secondary structure elements, or short linear motif (SLiMs), that undergo specific conformational transitions upon phosphorylation. Of importance, such conformational transition favors arrestin-2 binding. Hence, our results suggest a model in which the cellular signaling specificity of GPCRs is encoded in the phosphorylation-dependent structuration of the C-terminal regions, which will subsequently modulate arrestin conformation and therefore GPCR:arrestin signaling outcomes.

biophysics↗

bioRxiv2022

Statistical tests to detect differences between codon-specific Ramachandran plots

In their recent work, Rosenberg et al. [1] studied the dependence between the identity of synonymous codons and the distribution of the backbone dihedral angles of the translated amino acids. In the past, it has been shown that the use of synonymous codons is highly relevant in multiple biological processes including, among others, mRNA splicing, translational rates and protein folding [2, 3]. While the correlation between synonymous codons and secondary structure in translated proteins has been widely studied [4-6], Rosenberg et al. evaluated the effect of codon identity on a finer scale, analyzing whether the distribution of ({phi}, {psi}) dihedral angles within secondary structure elements is significantly altered when synonymous codons are used. Their conclusion, showing significant differences, particularly for amino acid residues involved in {beta}-strands, would represent a new paradigm for the role played by synonymous codons in defining protein structure. However, the statistical methodology used in that study was formally incorrect, casting doubt on the obtained results. Besides, it is based on density estimates that might be imprecise for small sample sizes, yielding misleading comparisons. These methodological errors are described in the following section. Then, using an appropriate methodology, we reanalyzed the data presented in [1]. Our results show that the influence of the codon on the distribution of the dihedral angles is statistically non-significant for all types of secondary structures, contradicting the conclusion by Rosenberg et al.. These results were corroborated by repeating the analysis on structures extracted from the AlphaFold Database [7, 8] for the same set of proteins, and shown to be robust with respect to the definition secondary structural classes and also when considering the nature of the neighbor residues. Overall, our observations demonstrate that the influence of the synonymous codons on the backbone dihedral angles can not be inferred with current data.

bioinformatics↗

bioRxiv2022

WASCO: A Wasserstein-based statistical tool to compare conformational ensembles of intrinsically disordered proteins

The structural investigation of intrinsically disordered proteins (IDPs) requires ensemble models describing the diversity of the conformational states of the molecule. Due to their probabilistic nature, there is a need for new paradigms that understand and treat IDPs from a purely statistical point of view, considering their conformational ensembles as well-defined probability distributions. In this work, we define a conformational ensemble as an ordered set of probability distributions and provide a suitable metric to detect differences between two given ensembles at the residue level, both locally and globally. The underlying geometry of the conformational space is properly integrated, being one ensemble characterized by a set of probability distributions supported on the three-dimensional Euclidean space (for global-scale comparisons) and on the two-dimensional flat torus (for local-scale comparisons). The inherent uncertainty of the data is also taken into account to provide finer estimations of the differences between ensembles. Additionally, an overall distance between ensembles is defined from the differences at the residue level. We illustrate the interest of the approach with several examples of applications for the comparison of conformational ensembles: (i) produced from molecular dynamics (MD) simulations using different force fields, and (ii) before and after refinement with experimental data. We also show the usefulness of the method to assess the convergence of MD simulations. The numerical tool has been implemented in Python through easy-to-use Jupyter Notebooks available at https://gitlab.laas.fr/moma/WASCO.

bioinformatics↗

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↗

bioRxiv2021

Conformational buffering underlies functional selection in intrinsically disordered protein regions

Many disordered proteins conserve essential functions in the face of extensive sequence variation. This makes it challenging to identify the forces responsible for functional selection. Viruses are robust model systems to investigate functional selection and they take advantage of protein disorder to acquire novel traits. Here, we combine structural and computational biophysics with evolutionary analysis to determine the molecular basis for functional selection in the intrinsically disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, triggering early S-phase entry and disrupting normal cellular proliferation. We show that the ability to outcompete host factors depends on the picomolar binding affinity of E1A for Rb, which is driven by two binding motifs tethered by a hypervariable disordered linker. Binding affinity is determined by the spatial dimensions of the linker, which constrain the relative position of the two binding motifs. Despite substantial sequence variation across evolution, the linker dimensions are finely optimized through compensatory changes in amino acid sequence and sequence length, leading to conserved linker dimensions and maximal affinity. We refer to the mechanism that conserves spatial dimensions despite large-scale variations in sequence as conformational buffering. Conformational buffering explains how variable disordered proteins encode functions and could be a general mechanism for functional selection within disordered protein regions.

biophysics↗

bioRxiv2021

Molecular and cellular insight into Escherichia coli SslE and its role during biofilm maturation

Escherichia coli is a Gram-negative bacterium that colonizes the human intestine and virulent strains can cause severe diarrhoeal and extraintestinal diseases. The protein SslE is secreted by a range of pathogenic and some commensal E. coli strains. It can degrade mucins in the intestine, promotes biofilm maturation and it is a major determinant of infection in virulent strains, although how it carries out these functions is not well understood. Here we examine SslE from the E. coli Waksman and H10407 strains and reveal that it has a novel and dynamic structure. In response to acidification within mature biofilms we show how SslE forms a unique functional aggregate that interacts with cellulose and regulates the distribution of exopolysaccharides in macrocolony biofilms. Our data indicates that the spatial organization of SslE polymers and local pH are critical for biofilm maturation and SslE is a key factor that drives persistence of SslE-secreting bacteria during acidic stress.

microbiology↗

bioRxiv2020

Structural insights into the cooperative interaction of the intrinsically disordered co-activator TIF2 with retinoic acid receptor heterodimer (RXR/RAR)

Retinoic acid receptors (RARs) and retinoid X receptors (RXRs) form heterodimers that activate target gene transcription by recruiting co-activator complexes in response to ligand binding. The nuclear receptor (NR) co-activator TIF2 mediates this recruitment by interacting with the ligand-binding domain (LBD) of NRs trough the nuclear receptor interaction domain (TIF2NRID) containing three highly conserved -helical LxxLL motifs (NR-boxes). The precise binding mode of this domain to RXR/RAR is not clear due to the disordered nature of TIF2. Here we present the structural characterization of TIF2NRID by integrating several experimental (NMR, SAXS, CD, SEC-MALS) and computational data. Collectively, the data are in agreement with a largely disordered protein with partially structured regions, including the NR-boxes and their flanking regions, which are evolutionary conserved. NMR and X-ray crystallographic data on TIF2NRID in complex with RXR/RAR reveal a cooperative binding of the three NR-boxes as well as an active role of their flanking regions in the interaction.

biophysics↗

bioRxiv2020

Multivalent interactions between eIF4G1, Pub1 and Pab1 drive the formation of protein condensates

Yeast eIF4G1 interacts with RNA binding proteins (RBPs) like Pab1 and Pub1 affecting its function in translation initiation and stress granules formation. We present an NMR and SAXS study of the intrinsically disordered region of eIF4G1, eIF4G11-249, and its interactions with Pub1 and Pab1. The conformational ensemble of eIF4G11-249 shows an -helix within the BOX3 conserved element and a dynamic network of fuzzy {pi}-{pi} and {pi}-cation interactions involving arginine and aromatic residues. The Pab1 RRM2 domain interacts with eIF4G1 BOX3, the canonical interaction site, but also with BOX2, a conserved element of unknown function to date. In contrast, the Pub1 RRM3 domain interacts with the RNA1-1 and BOX1 regions of eIF4G1. Mixtures of Pub1, Pab1 and eIF4G1 form micrometer-size protein condensates that require the presence of the eIF4G1 BOX1 element. These homotypic interactions suggest a double key mechanism of eIF4G1 regulation, important for understanding the architecture of stress granule cores.

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↗