RESUMEN
Nucleobases such as inosine have been extensively utilized to map direct contacts by proteins in the DNA groove. Their deployment as targeted probes of dynamics and hydration, which are dominant thermodynamic drivers of affinity and specificity, has been limited by a paucity of suitable experimental models. We report a joint crystallographic, thermodynamic, and computational study of the bidentate complex of the arginine side chain with a Watson-Crick guanine (Arg×GC), a highly specific configuration adopted by major transcription factors throughout the eukaryotic branches in the Tree of Life. Using the ETS-family factor PU.1 as a high-resolution structural framework, inosine substitution for guanine resulted in a sharp dissection of conformational dynamics and hydration and elucidated their role in the DNA specificity of PU.1. Our work suggests an under-exploited utility of modified nucleobases in untangling the structural thermodynamics of interactions, such as the Arg×GC motif, where direct and indirect readout are tightly integrated.
Asunto(s)
Proteínas Proto-Oncogénicas , Factores de Transcripción , Factores de Transcripción/metabolismo , Sitios de Unión , Unión Proteica , Proteínas Proto-Oncogénicas/química , Termodinámica , ADN/metabolismo , Guanina , Inosina/metabolismo , Conformación de Ácido NucleicoRESUMEN
Fixed-charge (non-polarizable) forcefields are accurate and computationally efficient tools for modeling the molecular dynamics of nucleic acid polymers, particularly DNA, well into the µs timescale. The continued utility of these forcefields depends in part on expanding the residue set in step with advancing nucleic acid chemistry and biology. A key step in parameterizing new residues is charge derivation which is self-consistent with the existing residues. As atomic charges are derived by fitting against molecular electrostatic potentials, appropriate structural models are critical. Benchmarking against the existing charge set used in current AMBER nucleic acid forcefields, we report that quantum mechanical models of deoxynucleosides, even at a high level of theory, are not optimal structures for charge derivation. Instead, structures from molecular mechanics minimization yield charges with up to 6-fold lower RMS deviation from the published values, due to the choice of such an approach in the derivation of the original charge set. We present a contemporary protocol for rendering self-consistent charges as well as optimized charges for a panel of nine non-canonical residues that will permit comparison with literature as well as studying the dynamics of novel DNA polymers.
RESUMEN
Somatic mutations in DNA-binding sites for CCCTC-binding factor (CTCF) are significantly elevated in many cancers. Prior analysis has suggested that elevated mutation rates at CTCF-binding sites in skin cancers are a consequence of the CTCF-cohesin complex inhibiting repair of UV damage. Here, we show that CTCF binding modulates the formation of UV damage to induce mutation hot spots. Analysis of genome-wide CPD-seq data in UV-irradiated human cells indicates that formation of UV-induced cyclobutane pyrimidine dimers (CPDs) is primarily suppressed by CTCF binding but elevated at specific locations within the CTCF motif. Locations of CPD hot spots in the CTCF-binding motif coincide with mutation hot spots in melanoma. A similar pattern of damage formation is observed at CTCF-binding sites in vitro, indicating that UV damage modulation is a direct consequence of CTCF binding. We show that CTCF interacts with binding sites containing UV damage and inhibits repair by a model repair enzyme in vitro. Structural analysis and molecular dynamic simulations reveal the molecular mechanism for how CTCF binding modulates CPD formation.
Asunto(s)
Factor de Unión a CCCTC/química , Reparación del ADN , Melanoma/genética , Proteínas Serina-Treonina Quinasas/química , Dímeros de Pirimidina/efectos de la radiación , Neoplasias Cutáneas/genética , Sitios de Unión , Unión Competitiva , Factor de Unión a CCCTC/genética , Factor de Unión a CCCTC/metabolismo , Línea Celular Tumoral , Daño del ADN , Expresión Génica , Humanos , Melanoma/metabolismo , Melanoma/patología , Simulación de Dinámica Molecular , Mutación , Unión Proteica , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Dímeros de Pirimidina/biosíntesis , Dímeros de Pirimidina/química , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Rayos UltravioletaRESUMEN
The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro-B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro- to pre-B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1's critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.
Asunto(s)
Agammaglobulinemia/genética , Cromatina/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Adolescente , Adulto , Linfocitos B/fisiología , Diferenciación Celular/genética , Línea Celular , Niño , Preescolar , Células Dendríticas/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/genética , Células HEK293 , Hematopoyesis/genética , Células Madre Hematopoyéticas/fisiología , Humanos , Lactante , Linfopoyesis/genética , Masculino , Mutación/genética , Células Precursoras de Linfocitos B/fisiología , Células Madre/fisiología , Adulto JovenRESUMEN
Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/metabolismo , Multimerización de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , ADN/metabolismo , Retroalimentación Fisiológica , Humanos , Proteínas Intrínsecamente Desordenadas/química , Mutación/genética , Conformación Proteica , Dominios Proteicos , Estabilidad Proteica , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Espectroscopía de Protones por Resonancia Magnética , Electricidad Estática , Transactivadores/química , Transactivadores/genética , Activación TranscripcionalRESUMEN
Functional evidence increasingly implicates low-affinity DNA recognition by transcription factors as a general mechanism for the spatiotemporal control of developmental genes. Although the DNA sequence requirements for affinity are well-defined, the dynamic mechanisms that execute cognate recognition are much less resolved. To address this gap, here we examined ETS1, a paradigm developmental transcription factor, as a model for which cognate discrimination remains enigmatic. Using molecular dynamics simulations, we interrogated the DNA-binding domain of murine ETS1 alone and when bound to high-and low-affinity cognate sites or to nonspecific DNA. The results of our analyses revealed collective backbone and side-chain motions that distinguished cognate versus nonspecific as well as high- versus low-affinity cognate DNA binding. Combined with binding experiments with site-directed ETS1 mutants, the molecular dynamics data disclosed a triad of residues that respond specifically to low-affinity cognate DNA. We found that a DNA-contacting residue (Gln-336) specifically recognizes low-affinity DNA and triggers the loss of a distal salt bridge (Glu-343/Arg-378) via a large side-chain motion that compromises the hydrophobic packing of two core helices. As an intact Glu-343/Arg-378 bridge is the default state in unbound ETS1 and maintained in high-affinity and nonspecific complexes, the low-affinity complex represents a unique conformational adaptation to the suboptimization of developmental enhancers.
Asunto(s)
ADN/química , ADN/metabolismo , Proteína Proto-Oncogénica c-ets-1/química , Proteína Proto-Oncogénica c-ets-1/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Conformación ProteicaRESUMEN
The ends of nucleic acids oligomers alter the statistics of interior nonspecific ligand binding and act as binding sites with altered properties. While the former aspect of "end effects" has received much theoretical attention in the literature, the physical nature of end-binding, and hence its potential impact on a wide range of studies with oligomers, remains poorly known. Here, we report for the first time end-binding to DNA using a model helix-turn-helix motif, the DNA-binding domain of ETV6, as a function of DNA sequence length. Spectral analysis of ETV6 intrinsic tryptophan fluorescence by singular value decomposition showed that end-binding to nonspecific fragments was negligible at >0.2 kbp and accumulated to 8% of total binding to 23-bp oligomers. The affinity for end-binding was insensitive to salt but tracked the affinity of interior binding, suggesting translocation from interior sites rather than free solution as its mechanism. As the presence of a cognate site in the 23-bp oligomer suppressed end-binding, neglect of end-binding to the short cognate DNA does not introduce significant error. However, the same applies to nonspecific DNA only if longer fragments (>0.2 kbp) are used.
Asunto(s)
ADN/química , Nucleoproteínas/química , Sitios de Unión , Modelos MolecularesRESUMEN
Hydration of interfaces is a major determinant of target specificity in protein/DNA interactions. Interfacial hydration is a highly variable feature in DNA recognition by ETS transcription factors and functionally relates to cellular responses to osmotic stress. To understand how hydration is mediated in the conserved ETS/DNA binding interface, secondary structures comprising the DNA contact surface of the strongly hydrated ETS member PU.1 were substituted, one at a time, with corresponding elements from its sparsely hydrated relative Ets-1. The resultant PU.1/Ets-1 chimeras exhibited variably reduced sensitivity to osmotic pressure, indicative of a distributed pattern of interfacial hydration in wildt-ype PU.1. With the exception of the recognition helix H3, the chimeras retained substantially high affinities. Ets-1 residues could therefore offset the loss of favorable hydration contributions in PU.1 via low-water interactions, but at the cost of decreased selectivity at base positions flanking the 5'-GGA-3' core consensus. Substitutions within H3 alone, which contacts the core consensus, impaired binding affinity and PU.1 transactivation in accordance with the evolutionary separation of the chimeric residues involved. The combined biophysical, bioinformatics and functional data therefore supports hydration as an evolved specificity determinant that endows PU.1 with more stringent sequence selection over its ancestral relative Ets-1.