RESUMEN
A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile, YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σE and σK and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B. subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabGC207A, both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σK-dependent genes, cotA, coding for a coat protein, and cdeM, coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B. subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.
Asunto(s)
Clostridioides difficile , Péptido Hidrolasas , Humanos , Péptido Hidrolasas/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Clostridioides , Esporas Bacterianas/metabolismo , Factores de Transcripción/metabolismo , Endopeptidasas/metabolismo , Proteínas Bacterianas/metabolismo , Bacillus subtilis/metabolismoRESUMEN
The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.
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Bases de Datos de Proteínas , Proteínas Intrínsecamente Desordenadas/química , Humanos , Motor de Búsqueda , Proteína p53 Supresora de Tumor/químicaRESUMEN
In many cases protein assemblies are stabilized by covalent bonds, one example of which is the formation of intra- or intermolecular ε-(γ-glutamyl)lysil cross-links catalyzed by transglutaminases (TGases). Because of the potential for unwanted cross-linking reactions, the activities of many TGases have been shown to be tightly controlled. Bacterial endospores are highly resilient cells in part because they are surrounded by a complex protein coat. Proteins in the coat that surrounds Bacillus subtilis endospores are crosslinked by a TGase (Tgl). Unlike other TGases, however, Tgl is produced in an active form, and efficiently catalyzes amine incorporation and protein cross-linking in vitro with no known additional requirements. The absence of regulatory factors raises questions as to how the activity of Tgl is controlled during spore coat assembly. Here, we show that substrates assembled onto the spore coat prior to Tgl production govern the localization of Tgl to the surface of the developing spore. We also show that Tgl residues important for substrate recognition are crucial for its localization. We identified the glutamyl (Q) and lysil (K) substrate docking sites and we show that residues on the Q side of Tgl are more important for the assembly of Tgl than those on the K side. Thus, the first step in the reaction cycle, the interaction with Q-substrates and formation of an acyl-enzyme intermediate, is also the determinant step in the localization of Tgl. Consistent with the idea that Tg exerts a "spotwelding" activity, cross-linking pre-formed assemblies, we show that C30 is an oblong hexamer in solution that is cross-linked in vitro into high molecular weight forms. Moreover, during the reaction, Tgl becomes part of the cross-linked products. We suggest that the dependency of Tgl on its substrates is used to accurately control the time, location and extent of the enzyme´s activity, directed at the covalent fortification of pre-assembled complexes at the surface of the developing spore.
Asunto(s)
Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Pared Celular/enzimología , Regulación Bacteriana de la Expresión Génica , Esporas Bacterianas/genética , Esporas Bacterianas/metabolismo , Transglutaminasas/metabolismo , Genes Reporteros , Modelos Moleculares , Conformación Molecular , Relación Estructura-Actividad , Especificidad por Sustrato , Transglutaminasas/química , Transglutaminasas/genéticaRESUMEN
Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations concentrate at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple molecular dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by small-angle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806-epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein.
Asunto(s)
Genes erbB-1 , Glioblastoma/genética , Epítopos , Células HEK293 , Humanos , Mutación MissenseRESUMEN
The intrinsically disordered p15PAF regulates DNA replication and repair when interacting with the Proliferating Cell Nuclear Antigen (PCNA) sliding clamp. As many interactions between disordered proteins and globular partners involved in signaling and regulation, the complex between p15PAF and trimeric PCNA is of low affinity, forming a transient complex that is difficult to characterize at a structural level due to its inherent polydispersity. We have determined the structure, conformational fluctuations, and relative population of the five species that coexist in solution by combining small-angle X-ray scattering (SAXS) with molecular modelling. By using explicit ensemble descriptions for the individual species, built using integrative approaches and molecular dynamics (MD) simulations, we collectively interpreted multiple SAXS profiles as population-weighted thermodynamic mixtures. The analysis demonstrates that the N-terminus of p15PAF penetrates the PCNA ring and emerges on the back face. This observation substantiates the role of p15PAF as a drag regulating PCNA processivity during DNA repair. Our study reveals the power of ensemble-based approaches to decode structural, dynamic, and thermodynamic information from SAXS data. This strategy paves the way for deciphering the structural bases of flexible, transient and multivalent macromolecular assemblies involved in pivotal biological processes.
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Proteínas Portadoras/química , Antígeno Nuclear de Célula en Proliferación/química , Proteínas de Unión al ADN , Humanos , Proteínas Intrínsecamente Desordenadas/química , Simulación de Dinámica Molecular , Complejos Multiproteicos/química , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Intrinsically Disordered Proteins (IDPs) are fundamental actors of biological processes. Their inherent plasticity facilitates very specialized tasks in cell regulation and signalling, and their malfunction is linked to severe pathologies. Understanding the functional role of disorder requires the structural characterization of IDPs and the complexes they form. Small-angle Scattering of X-rays (SAXS) and Neutrons (SANS) have notably contributed to this structural understanding. In this review we summarize the most relevant developments in the field of SAS studies of disordered proteins. Emphasis is given to ensemble methods and how SAS data can be combined with computational approaches or other biophysical information such as NMR. The unique capabilities of SAS enable its application to extremely challenging disordered systems such as low-complexity regions, amyloidogenic proteins and transient biomolecular complexes. This reinforces the fundamental role of SAS in the structural and dynamic characterization of this elusive family of proteins.
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Proteínas Amiloidogénicas/ultraestructura , Proteínas Intrínsecamente Desordenadas/ultraestructura , Modelos Moleculares , Dispersión del Ángulo Pequeño , Proteínas Amiloidogénicas/química , Simulación por Computador , Humanos , Proteínas Intrínsecamente Desordenadas/química , Difracción de Neutrones/instrumentación , Difracción de Neutrones/métodos , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Sincrotrones/instrumentación , Difracción de Rayos X/instrumentación , Difracción de Rayos X/métodosRESUMEN
The Hha/YmoA nucleoid-associated proteins help selectively silence horizontally acquired genetic material, including pathogenicity and antibiotic resistance genes and their maintenance in the absence of selective pressure. Members of the Hha family contribute to gene silencing by binding to the N-terminal dimerization domain of H-NS and modifying its selectivity. Hha-like proteins and the H-NS N-terminal domain are unusually rich in charged residues, and their interaction is mostly electrostatic-driven but, nonetheless, highly selective. The NMR-based structural model of the complex between Hha/YmoA and the H-NS N-terminal dimerization domain reveals that the origin of the selectivity is the formation of a three-protein charge zipper with interdigitated complementary charged residues from Hha and the two units of the H-NS dimer. The free form of YmoA shows collective microsecond-millisecond dynamics that can by measured by NMR relaxation dispersion experiments and shows a linear dependence with the salt concentration. The number of residues sensing the collective dynamics and the population of the minor form increased in the presence of H-NS. Additionally, a single residue mutation in YmoA (D43N) abolished H-NS binding and the dynamics of the apo-form, suggesting the dynamics and binding are functionally related.
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Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Enterobacteriaceae/genética , Enterobacteriaceae/metabolismo , Regulación Bacteriana de la Expresión Génica , Transferencia de Gen Horizontal , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas de Unión al ADN/química , Infecciones por Enterobacteriaceae/microbiología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Mapas de Interacción de Proteínas , Multimerización de Proteína , Electricidad EstáticaRESUMEN
We present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15(PAF), showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15(PAF) is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15(PAF) gene is overexpressed in several types of human cancer. The nearly complete NMR backbone assignment of p15(PAF) allowed us to measure 86 N-H(N) residual dipolar couplings. Our residual dipolar coupling analysis reveals nonrandom conformational preferences in distinct regions, including the proliferating-cell-nuclear-antigen-interacting protein motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that targets p15(PAF) for degradation). In accordance with these findings, analysis of the (15)N R2 relaxation rates shows a relatively reduced mobility for the residues in these regions. The agreement between the experimental small angle x-ray scattering curve of p15(PAF) and that computed from a statistical coil ensemble corrected for the presence of local secondary structural elements further validates our structural model for p15(PAF). The coincidence of these transiently structured regions with protein-protein interaction and posttranslational modification sites suggests a possible role for these structures as molecular recognition elements for p15(PAF).
Asunto(s)
Proteínas Portadoras/química , Secuencia de Aminoácidos , Sitios de Unión , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Humanos , Datos de Secuencia Molecular , Unión ProteicaRESUMEN
Ler is a DNA-binding, oligomerizable protein that regulates pathogenicity islands in enterohemorrhagic and enteropathogenic Escherichia coli strains. Ler counteracts the transcriptional silencing effect of H-NS, another oligomerizable nucleoid-associated protein. We studied the oligomerization of Ler in the absence and presence of DNA by atomic force microscopy. Ler forms compact particles with a multimodal size distribution corresponding to multiples of 3-5 units of Ler. DNA wraps around Ler particles that contain more than 15-16 Ler monomers. The resulting shortening of the DNA contour length is in agreement with previous measurements of the length of DNA protected by Ler in footprinting assays. We propose that the repetition unit corresponds to the number of monomers per turn of a tight helical Ler oligomer. While the repressor (H-NS) and anti-repressor (Ler) have similar DNA-binding domains, their oligomerization domains are unrelated. We suggest that the different oligomerization behavior of the two proteins explains the opposite results of their interaction with the same or proximal regions of DNA.
Asunto(s)
ADN Bacteriano/ultraestructura , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Transactivadores/química , ADN Bacteriano/química , Proteínas de Unión al ADN/ultraestructura , Escherichia coli Enterohemorrágica/genética , Escherichia coli Enterohemorrágica/patogenicidad , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/ultraestructura , Microscopía de Fuerza Atómica , Multimerización de Proteína , Transactivadores/ultraestructuraRESUMEN
Ribonucleases are in charge of the processing, degradation and quality control of all cellular transcripts, which makes them crucial factors in RNA regulation. This post-transcriptional regulation allows bacteria to promptly react to different stress conditions and growth phase transitions, and also to produce the required virulence factors in pathogenic bacteria. Campylobacter jejuni is the main responsible for human gastroenteritis in the world. In this foodborne pathogen, exoribonuclease PNPase (CjPNP) is essential for low-temperature cell survival, affects the synthesis of proteins involved in virulence and has an important role in swimming, cell adhesion/invasion ability, and chick colonization. Here we report the crystallographic structure of CjPNP, complemented with SAXS, which confirms the characteristic doughnut-shaped trimeric arrangement and evaluates domain arrangement and flexibility. Mutations in highly conserved residues were constructed to access their role in RNA degradation and polymerization. Surprisingly, we found two mutations that altered CjPNP into a protein that is only capable of degrading RNA even in conditions that favour polymerization. These findings will be important to develop new strategies to combat C. jejuni infections.
Asunto(s)
Campylobacter jejuni , Polirribonucleótido Nucleotidiltransferasa , Humanos , Virulencia , Polirribonucleótido Nucleotidiltransferasa/genética , Polirribonucleótido Nucleotidiltransferasa/química , Polirribonucleótido Nucleotidiltransferasa/metabolismo , Dispersión del Ángulo Pequeño , Difracción de Rayos X , Endorribonucleasas , ARN , Exorribonucleasas/metabolismo , Ribonucleasa PancreáticaRESUMEN
The translocated intimin receptor (Tir) is an essential type III secretion system (T3SS) effector of attaching and effacing pathogens contributing to the global foodborne disease burden. Tir acts as a cell-surface receptor in host cells, rewiring intracellular processes by targeting multiple host proteins. We investigated the molecular basis for Tir's binding diversity in signalling, finding that Tir is a disordered protein with host-like binding motifs. Unexpectedly, also are several other T3SS effectors. By an integrative approach, we reveal that Tir dimerises via an antiparallel OB-fold within a highly disordered N-terminal cytosolic domain. Also, it has a long disordered C-terminal cytosolic domain partially structured at host-like motifs that bind lipids. Membrane affinity depends on lipid composition and phosphorylation, highlighting a previously unrecognised host interaction impacting Tir-induced actin polymerisation and cell death. Furthermore, multi-site tyrosine phosphorylation enables Tir to engage host SH2 domains in a multivalent fuzzy complex, consistent with Tir's scaffolding role and binding promiscuity. Our findings provide insights into the intracellular Tir domains, highlighting the ability of T3SS effectors to exploit host-like protein disorder as a strategy for host evasion.
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Proteínas de Escherichia coli , Proteínas de Escherichia coli/metabolismo , Proteínas Portadoras , Receptores de Superficie Celular/metabolismoRESUMEN
Ler, a member of the H-NS protein family, is the master regulator of the LEE pathogenicity island in virulent Escherichia coli strains. Here, we determined the structure of a complex between the DNA-binding domain of Ler (CT-Ler) and a 15-mer DNA duplex. CT-Ler recognizes a preexisting structural pattern in the DNA minor groove formed by two consecutive regions which are narrower and wider, respectively, compared with standard B-DNA. The compressed region, associated with an AT-tract, is sensed by the side chain of Arg90, whose mutation abolishes the capacity of Ler to bind DNA. The expanded groove allows the approach of the loop in which Arg90 is located. This is the first report of an experimental structure of a DNA complex that includes a protein belonging to the H-NS family. The indirect readout mechanism not only explains the capacity of H-NS and other H-NS family members to modulate the expression of a large number of genes but also the origin of the specificity displayed by Ler. Our results point to a general mechanism by which horizontally acquired genes may be specifically recognized by members of the H-NS family.
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ADN Forma B/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiología , Escherichia coli/patogenicidad , Proteínas Fimbrias/fisiología , Transactivadores/metabolismo , Arginina/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli/química , Proteínas Fimbrias/química , Transferencia de Gen Horizontal , Islas Genómicas , Estructura Terciaria de Proteína , Transactivadores/químicaRESUMEN
Rhodopsin-1 (Rh1), the main photosensitive protein of Drosophila, is a seven-transmembrane domain protein, which is inserted co-translationally in the endoplasmic reticulum (ER) membrane. Biogenesis of Rh1 occurs in the ER, where various chaperones interact with Rh1 to aid in its folding and subsequent transport from the ER to the rhabdomere, the light-sensing organelle of the photoreceptors. Xport-A has been proposed as a chaperone/transport factor for Rh1, but the exact molecular mechanism for Xport-A activity upon Rh1 is unknown. Here, we propose a model where Xport-A functions as a chaperone during the biogenesis of Rh1 in the ER by stabilizing the first five transmembrane domains (TMDs) of Rh1.
RESUMEN
Laccases are in increasing demand as innovative solutions in the biorefinery fields. Here, we combine mutagenesis with structural, kinetic, and in silico analyses to characterize the molecular features that cause the evolution of a hyperthermostable metallo-oxidase from the multicopper oxidase family into a laccase (k cat 273 s-1 for a bulky aromatic substrate). We show that six mutations scattered across the enzyme collectively modulate dynamics to improve the binding and catalysis of a bulky aromatic substrate. The replacement of residues during the early stages of evolution is a stepping stone for altering the shape and size of substrate-binding sites. Binding sites are then fine-tuned through high-order epistasis interactions by inserting distal mutations during later stages of evolution. Allosterically coupled, long-range dynamic networks favor catalytically competent conformational states that are more suitable for recognizing and stabilizing the aromatic substrate. This work provides mechanistic insight into enzymatic and evolutionary molecular mechanisms and spots the importance of iterative experimental and computational analyses to understand local-to-global changes.
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Bacteriophages (phages) and other viruses are extremely efficient in packing their genetic information, with several described cases of overlapping genes encoded in different open reading frames (ORFs). While less frequently reported, specific cases exist in which two overlapping ORFs are in frame and share the stop codon. Here, we studied the occurrence of this genetic arrangement in endolysins, the phage enzymes that cut the bacterial cell wall peptidoglycan to release the virion progeny. After screening over 3,000 endolysin sequences of phages infecting Gram-positive bacteria, we found evidence that this coding strategy is frequent in endolysin genes. Our bioinformatics predictions were experimentally validated by demonstrating that two polypeptides are indeed produced from these genes. Additionally, we show that in some cases the two polypeptides need to interact and multimerize to generate the active endolysin. By studying in detail one selected example, we uncovered a heteromeric endolysin with a 1:5 subunit stoichiometry that has never been described before. Hence, we conclude that the occurrence of endolysin genes encoding two polypeptide isoforms by in-frame overlapping ORFs, as well as their organization as enzymatic complexes, appears more common than previously thought, therefore challenging the established view of endolysins being mostly formed by single, monomeric polypeptide chains. IMPORTANCE Bacteriophages use endolysins to cleave the host bacteria cell wall, a crucial event underlying cell lysis for virion progeny release. These bacteriolytic enzymes are generally thought to work as single, monomeric polypeptides, but a few examples have been described in which a single gene produces two endolysin isoforms. These are encoded by two in-frame overlapping ORFs, with a shorter ORF being defined by an internal translation start site. This work shows evidence that this endolysin coding strategy is frequent in phages infecting Gram-positive bacteria, and not just an eccentricity of a few phages. In one example studied in detail, we show that the two isoforms are inactive until they assemble to generate a multimeric active endolysin, with a 1:5 subunit stoichiometry never described before. This study challenges the established view of endolysins, with possible implications in their current exploration and design as alternative antibacterials.
Asunto(s)
Bacteriófagos , Bacterias , Bacteriófagos/genética , Pared Celular , Endopeptidasas/química , Endopeptidasas/genética , PeptidoglicanoRESUMEN
Arrestin-dependent pathways are a central component of G protein-coupled receptor (GPCRs) signaling. However, the molecular processes regulating arrestin binding are to be further illuminated, in particular with regard to the structural impact of GPCR C-terminal disordered regions. Here, we used an integrated biophysical strategy to describe the basal conformations of the C-terminal domains of three class A GPCRs, the vasopressin V2 receptor (V2R), the growth hormone secretagogue or ghrelin receptor type 1a (GHSR) and the ß2-adernergic receptor (ß2AR). By doing so, we revealed the presence of transient secondary structures in these regions that are potentially involved in the interaction with arrestin. These secondary structure elements differ from those described in the literature in interaction with arrestin. This suggests a mechanism where the secondary structure conformational preferences in the C-terminal regions of GPCRs could be a central feature for optimizing arrestins recognition.
Asunto(s)
Arrestina , Arrestinas , Arrestina/metabolismo , Arrestinas/metabolismo , Estructura Secundaria de Proteína , Receptores Acoplados a Proteínas G/metabolismoRESUMEN
SMAD transcription factors, the main effectors of the TGFß (transforming growth factor ß) network, have a mixed architecture of globular domains and flexible linkers. Such a complicated architecture precluded the description of their full-length (FL) structure for many years. In this study, we unravel the structures of SMAD4 and SMAD2 proteins through an integrative approach combining Small-angle X-ray scattering, Nuclear Magnetic Resonance spectroscopy, X-ray, and computational modeling. We show that both proteins populate ensembles of conformations, with the globular domains tethered by disordered and flexible linkers, which defines a new dimension of regulation. The flexibility of the linkers facilitates DNA and protein binding and modulates the protein structure. Yet, SMAD4FL is monomeric, whereas SMAD2FL is in different monomer-dimer-trimer states, driven by interactions of the MH2 domains. Dimers are present regardless of the SMAD2FL activation state and concentration. Finally, we propose that SMAD2FL dimers are key building blocks for the quaternary structures of SMAD complexes.
RESUMEN
Smad transcription factors are the main downstream effectors of the Transforming growth factor ß superfamily (TGFß) signalling network. The DNA complexes determined here by X-ray crystallography for the Bone Morphogenetic Proteins (BMP) activated Smad5 and Smad8 proteins reveal that all MH1 domains bind [GGC(GC)|(CG)] motifs similarly, although TGFß-activated Smad2/3 and Smad4 MH1 domains bind as monomers whereas Smad1/5/8 form helix-swapped dimers. Dimers and monomers are also present in solution, as revealed by NMR. To decipher the characteristics that defined these dimers, we designed chimeric MH1 domains and characterized them using X-ray crystallography. We found that swapping the loop1 between TGFß- and BMP- activated MH1 domains switches the dimer/monomer propensities. When we scanned the distribution of Smad-bound motifs in ChIP-Seq peaks (Chromatin immunoprecipitation followed by high-throughput sequencing) in Smad-responsive genes, we observed specific site clustering and spacing depending on whether the peaks correspond to BMP- or TGFß-responsive genes. We also identified significant correlations between site distribution and monomer or dimer propensities. We propose that the MH1 monomer or dimer propensity of Smads contributes to the distinct motif selection genome-wide and together with the MH2 domain association, help define the composition of R-Smad/Smad4 trimeric complexes.
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Most model bacteria have been domesticated in laboratory conditions. Yet, the tempo with which a natural isolate diverges from its ancestral phenotype under domestication to a novel laboratory environment is poorly understood. Such knowledge, however is essential to understanding the rate of evolution, the time scale over 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 to 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 target for mutations during domestication and underscores the importance of performing careful and extremely short-term propagations of natural isolates to conserve the traits encoded in their original genomes.
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Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Mutación , Secuenciación Completa del Genoma/métodos , Adaptación Fisiológica , Animales , Bacillus subtilis/genética , Bacillus subtilis/aislamiento & purificación , Proteínas Bacterianas/química , Biopelículas/crecimiento & desarrollo , Evolución Molecular , Regulación Bacteriana de la Expresión Génica , Homeostasis , Ratones , Modelos Moleculares , Fenotipo , Conformación Proteica , Células RAW 264.7RESUMEN
In its unliganded form, the retinoic acid receptor (RAR) in heterodimer with the retinoid X receptor (RXR) exerts a strong repressive activity facilitated by the recruitment of transcriptional corepressors in the promoter region of target genes. By integrating complementary structural, biophysical, and computational information, we demonstrate that intrinsic disorder is a required feature for the precise regulation of RAR activity. We show that structural dynamics of RAR and RXR H12 regions is an essential mechanism for RAR regulation. Unexpectedly we found that, while mainly disordered, the corepressor N-CoR presents evolutionary conserved structured regions involved in transient intramolecular contacts. In the presence of RXR/RAR, N-CoR exploits its multivalency to form a cooperative multisite complex that displays equilibrium between different conformational states that can be tuned by cognate ligands and receptor mutations. This equilibrium is key to preserving the repressive basal state while allowing the conversion to a transcriptionally active form.