Biology subjects

Cordeiro, T. N.

Publications and source records attributed to Cordeiro, T. N..

8 recordsLinked to original sources

bioRxiv2025

Ensemblify: a user-friendly tool for generating ensembles of intrinsically disordered regions of AlphaFold and user-defined models

MotivationIntrinsically disordered proteins (IDPs) and regions (IDRs) challenge structural characterization due to their dynamic conformational ensembles and lack of stable structure. Existing computational approaches for modelling these ensembles are either computationally intensive, limited in flexibility, or inaccessible to non-experts, especially when dealing with multi-domain or multi-chain proteins. ResultsWe present Ensemblify, an open-source, user-friendly Python package for generating and analyzing conformational ensembles of IDPs/IDRs. Ensemblify uses a Monte Carlo algorithm coupled with neighbour-aware sampling of dihedral angles from curated or user-defined fragment libraries to explore conformational space. It directly incorporates information from AlphaFolds confidence metrics as flexible energy restraints in PyRosetta to guide the sampling. It supports multi-chain and multi-domain proteins and can sample N-terminal, C-terminal, and inter-domain linkers while preserving folded regions. Ensemble quality can be validated and refined against experimental data such as SAXS via Bayesian/Maximum Entropy (BME) reweighting. Interactive dashboards provide in-depth structural analysis and comparison. Testing across 10 diverse proteins demonstrated Ensemblifys accuracy, flexibility, and ability to recover experimentally observed structural features. Incorporating AlphaFold confidence metrics shows potential to improve the ensemble-data agreement. AvailabilityEnsemblify is freely available at https://github.com/CordeiroLab/ensemblify, along with detailed installation instructions and usage tutorials. Ensemblify can be used for scripting through its Python API or directly through the provided command-line interface (CLI). Complete documentation is available within the source-code and CLI and on Ensemblifys official documentation page (https://ensemblify.readthedocs.io). Contacttiago.gomes@itqb.unl.pt, tiago.cordeiro@itqb.unl.pt Supplementary informationSupplementary data is available online.

bioinformatics↗

bioRxiv2024

Fbxo42 promotes the degradation of Ataxin-2 granules to trigger terminal Xbp1 signaling

The Unfolded Protein Response (UPR) is composed by homeostatic signaling pathways that are activated by the accumulation of misfolded proteins in the Endoplasmic Reticulum (ER), a condition known as ER stress. Prolonged ER stress and activation of the UPR causes cell death, by mechanisms that remain poorly understood. Here, we report that regulation of Ataxin-2 by Fbxo42 is a crucial step during UPR-induced cell death. From a genetic screen in Drosophila, we identified loss of function mutations in Fbxo42 that suppress cell death and retinal degeneration induced by the overexpression of Xbp1spliced, an important mediator of the UPR. We identified the RNA binding protein Ataxin-2 as a substrate of Fbxo42, which, as part of a Skp-A/Cullin-1 complex, promotes the ubiquitylation and degradation of Ataxin-2. Upon ER-stress, the mRNA of Xbp1 is not immediately translated but instead it is sequestered and stabilized in Ataxin-2 granules, until the Fbxo42 recruitment to these granules promotes the degradation of Ataxin-2, allowing the translation of Xbp1 mRNA at later stages of UPR activation. Our results identify Fbxo42-mediated degradation of Ataxin-2 as a key mechanism regulating the levels of Xbp1 mRNA and protein, to trigger cell death during the terminal stages of UPR activation.

cell biology↗

bioRxiv2022

Xport-A functions as a chaperone by stabilizing the first 5 transmembrane domains of Rhodopsin-1

Rhodopsin-1 (Rh1), the main photo-sensitive protein of Drosophila, is a seven transmembrane domain protein, which is inserted co-translationally in the endoplasmic reticulum (ER) membrane. Maturation of Rh1 occurs in the ER, where various chaperones interact with Rh1 to aid in its folding and subsequent transport in the secretory pathway. Xport-A has been shown to be a chaperone/ transport factor for Rh1, but the exact molecular mechanism for Xport-A activity upon Rh1 is not known. Here, based on computational predictions, we propose a model where Xport-A functions as a chaperone in the biosynthesis of Rh1 by stabilizing the first 5 transmembrane domains of Rh1, but not the full length Rh1 protein.

cell biology↗

bioRxiv2021

A pathogen-encoded signaling receptor mediating host-like interactions through intrinsic disorder

The translocated intimin receptor (Tir) is a central effector of Attaching and Effacing (A/E) pathogens responsible for worldwide foodborne disease cases. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes to assist infection by targeting multiple host proteins. We sought to understand the basis for Tir binding diversity in signaling. Here, we establish that Tir is a disordered protein with host-like binding motifs. A trait we find prevalent in several other effectors secreted by A/E bacteria. We disclose that Tir has a disordered C-terminal intracellular tail (C-Tir) with non-random structural preferences at phosphorylation sites, including host-like tyrosine-based motifs, with versatile lipid- and SH2 domain binding capability pre-phosphorylation. We show that multi-site tyrosine phosphorylation enables C-Tir to engage SH2 domains in a multivalent manner, consistent with Tirs scaffold/hub role for host proteins. Last, we uncover Tirs ability to dimerizes via its partially disordered N-terminal intracellular domain. Collectively, our findings provide an updated picture of Tirs intracellular side, highlighting its ability to mimic host disordered membrane receptors versatility as a molecular strategy for host evasion. SummaryTir is a cell-surface receptor secreted by life-threatening pathogens. Upon delivery into host cells, Tir inserts the host plasma membrane providing a means for these extracellular pathogens to control host intracellular processes. To prevent pathogens from relying on Tir, it is essential to understand its intracellular mechanics. This paper provides a coherent picture of the intracellular side of Tir, highlighting its ability to copycat the interactions of disordered intracellular domains of host immune receptors. This copycatting allows the bacterial pathogens to modulate critical host processes, allowing infection to spread further without triggering the immune system response. This work proposes that other bacterial secreted pathogenic proteins exploit intrinsic disorder to hijack human cells, suggesting a widespread host subversion mechanism.

biophysics↗

bioRxiv2021

Conformational landscape of full-length Smad proteins

Smad transcription factors, the main effectors of the TGF{beta} (transforming growth factor {beta}) network, have been shaped along the evolution of multicellular animals to regulate essential processes. Smad proteins have a mixed architecture of globular domains and flexible linkers and adopt distinct quaternary structures depending on their activation state and cellular context. Here we studied the structures of full-length Smad4 and Smad2 proteins through an integrative approach combining small-angle X-ray scattering and detailed atomic information obtained from Nuclear Magnetic Resonance spectroscopy, X-ray and molecular dynamic simulations. Both Smad4 and Smad2 populate ensembles of expanded/compact conformations, with the MH1 and MH2 domains tethered by intrinsically disordered linkers that provide conformational freedom to the proteins. In solution, Smad4 is monomeric, whereas Smad2 coexists as monomer-dimer-trimer association states, even without activation. Smad2 dimers, which were previously overlooked, are proposed as key building blocks that define the functional quaternary structures of Smad proteins. HighlightsO_LISolution ensembles of full-length Smad2 and Smad4 proteins C_LIO_LISmad4 populates dynamic extended and compact monomeric conformations C_LIO_LISmad2 dimers may be the intermediate state to form heterotrimers with Smad4 C_LIO_LINew integrative methodological strategy for examining multi-domain and flexible proteins C_LI

molecular biology↗

bioRxiv2019

Unveiling the dimer/monomer propensities of Smad MH1-DNA complexes

R-Smads are effectors of the transforming growth factor {beta} (TGF{beta}) superfamily and along with Smad4 form trimers to interact with other transcription factors and with DNA. The 5GC-DNA complexes determined here by X-ray crystallography for Smad5 and Smad8 proteins corroborate that all MH1 domains bind SBE and 5GC sites similarly, although Smad2/3/4 MH1 domains bind DNA as monomers whereas Smad1/5/8 form helix-swapped dimers. To examine the relevance of the dimerization phenomenon and to exclude a possible crystallography-induced dimeric state, we studied these MH1 domains in solution. The results show that Smad5/8 domains populate dimers and monomers in equilibrium, whereas their Smad2/3/4 counterparts adopt monomeric conformations. We also found that swapping the loop1 sequence between Smad5 and Smad3 results in the Smad5 chimera-DNA complex crystallizing as a monomer, revealing that the loop1 sequence determines the monomer/dimer propensity of Smad MH1-domains. We propose that the distinct MH1-dimerization status of TGF{beta} and BMP activated Smads influences the interaction with specific loci genome-wide by distinct R-Smad and Smad4 complexes. SignificanceTGF{beta}- and BMP-activated R-Smads were believed to have different preferences with respect to the recognition of DNA motifs and to respond to specific activation inputs. However, recent results indicate that several types of R-Smads can be activated by similar receptors and that all Smads might recognize various DNA motifs. These results pose new questions as to why different types of R-Smads have been conserved for more than 500 million years if they could have a redundant function. They also raise questions as to how different Smad complexes recognize specific clusters of DNA motifs genome-wide. Here, using structural biology approaches, we elucidate some of the rules that help define the dimers of Smad-DNA complexes and propose how dimers and monomers could influence the composition of Smad complexes, as well as the recognition of specific cis-regulatory elements genome-wide. HighlightsR-Smads and Smad4 interact with 5GC and GTCT sites using a conserved binding mode. Functional differences of TGF{beta}- and BMP-activated R-Smads are not exclusively related to DNA specificity. Dimer/monomer propensities are detected in solution and in the absence of DNA. Loop1 sequence determines the propensity of R-Smads to form monomers or dimers in complexes with DNA. Author ContributionsL.R., Z.K., and T.G. designed and performed most experiments and coordinated collaborations with other authors. L.R. and E.A. cloned, expressed and purified all proteins, L.R., R.F., C.T., N.M., and J.C performed EMSA experiments. L.R., E.A., T.G., T.C., P.M.M., and M.J.M. performed the SAXS and NMR measurements and analyzed the data. P.M.M. analyzed the clustering of DNA motifs in ChIP-Seq data. Z.K. B.B. and R.P. screened crystallization conditions, collected X-ray data and determined the structures. Z.K., R.F. R.P., T.G., J.A.M., and M.J.M. analyzed the structures. All authors contributed ideas to the project. M.J.M. and R.P. supervised the project. M.J.M. wrote the manuscript with contributions from all other authors. The authors declare no conflict of interest. Data DepositionNMR assignments and chemical shifts have been deposited in the Biological Magnetic Resonance Data Bank, BMRB entry 27548, and the Small-angle scattering data and models have been deposited in SASBDB, entries SASDE32 (Smad5) and SASDE42 (Smad8). Densities and coordinates have been deposited in the Protein Data Bank, entries 6FZS (Smad5), 6FZT (Smad8), 6TBZ (Smad5_3 chimera), 6TCE (Smad5_gly mutant). This article contains supporting information.

molecular biology↗

bioRxiv2019

Rampant loss of social traits during domestication of a wild Bacillus subtilis

Most well-studied bacteria have been domesticated to some extent. How fast can a natural isolate diverge from its ancestral phenotypes under domestication to a novel laboratory environment is poorly known. Yet such information is key to understand rates of evolution, the time scale at which a natural isolate can be propagated without loss of its natural adaptive traits and the reliability of experimental results across labs. Using experimental evolution, phenotypic assays and whole-genome sequencing, we show that within a week of propagation in a common laboratory environment, a natural isolate of Bacillus subtilis acquires mutations that cause changes in a multitude of traits. A single adaptive mutational step, in the gene coding for the transcriptional regulator DegU, impairs a DegU-dependent positive autoregulatory loop and leads to loss of robust biofilm architecture, impaired swarming motility, reduced secretion of exoproteases and changes in the dynamics of sporulation across environments. Importantly, domestication also resulted in improved survival when the bacteria face pressure from cells of the innate immune system. These results show that degU is a key target for mutations during domestication and also underscore the importance of performing careful and extremely short-term propagations of natural isolates to conserve the traits encoded in their original genomes. SummaryDomestication is the process by which organisms are selected to live in specific conditions and an important phenomenon that shapes the evolution and variation in many animals and plants. In microbes, domestication is also a key driver of adaptation. It can be beneficial, when improving microbes abilities that are important for biotechnology, but also problematic, especially when studying microbe-host interactions and the microbes natural behavior. Using a natural isolate of Bacillus subtilis, we determined the speed and genetic basis of microbial domestication using experimental evolution. Within one week of growth in the common laboratory media, mutations in the pleiotropic transcriptional regulator, DegU, emerge and spread in the populations. These lead to loss of social traits, increased resistance to bacteriophages and increased survival in the presence of macrophages. The data highlights the extreme caution that is needed when culturing natural microbial isolates and may help explain why some key microbial social traits and behaviors may differ between different laboratories, even when studying the same strains.

evolutionary biology↗

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↗