Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 31
Filtrar
1.
Cell ; 170(3): 457-469.e13, 2017 Jul 27.
Artículo en Inglés | MEDLINE | ID: mdl-28753425

RESUMEN

G protein-coupled receptors (GPCRs) mediate diverse signaling in part through interaction with arrestins, whose binding promotes receptor internalization and signaling through G protein-independent pathways. High-affinity arrestin binding requires receptor phosphorylation, often at the receptor's C-terminal tail. Here, we report an X-ray free electron laser (XFEL) crystal structure of the rhodopsin-arrestin complex, in which the phosphorylated C terminus of rhodopsin forms an extended intermolecular ß sheet with the N-terminal ß strands of arrestin. Phosphorylation was detected at rhodopsin C-terminal tail residues T336 and S338. These two phospho-residues, together with E341, form an extensive network of electrostatic interactions with three positively charged pockets in arrestin in a mode that resembles binding of the phosphorylated vasopressin-2 receptor tail to ß-arrestin-1. Based on these observations, we derived and validated a set of phosphorylation codes that serve as a common mechanism for phosphorylation-dependent recruitment of arrestins by GPCRs.


Asunto(s)
Arrestinas/química , Rodopsina/química , Secuencia de Aminoácidos , Animales , Arrestinas/metabolismo , Cromatografía Liquida , Humanos , Ratones , Modelos Moleculares , Fosforilación , Ratas , Rodopsina/metabolismo , Alineación de Secuencia , Espectrometría de Masas en Tándem , Rayos X
2.
Cell ; 157(7): 1685-97, 2014 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-24949977

RESUMEN

The glucocorticoid receptor (GR), like many signaling proteins, depends on the Hsp90 molecular chaperone for in vivo function. Although Hsp90 is required for ligand binding in vivo, purified apo GR is capable of binding ligand with no enhancement from Hsp90. We reveal that Hsp70, known to facilitate client delivery to Hsp90, inactivates GR through partial unfolding, whereas Hsp90 reverses this inactivation. Full recovery of ligand binding requires ATP hydrolysis on Hsp90 and the Hop and p23 cochaperones. Surprisingly, Hsp90 ATP hydrolysis appears to regulate client transfer from Hsp70, likely through a coupling of the two chaperone's ATP cycles. Such coupling is embodied in contacts between Hsp90 and Hsp70 in the GR:Hsp70:Hsp90:Hop complex imaged by cryoelectron microscopy. Whereas GR released from Hsp70 is aggregation prone, release from Hsp90 protects GR from aggregation and enhances its ligand affinity. Together, this illustrates how coordinated chaperone interactions can enhance stability, function, and regulation.


Asunto(s)
Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína
3.
Nat Chem Biol ; 13(1): 111-118, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27870835

RESUMEN

Resistance to endocrine therapies remains a major clinical problem for the treatment of estrogen receptor-α (ERα)-positive breast cancer. On-target side effects limit therapeutic compliance and use for chemoprevention, highlighting an unmet need for new therapies. Here we present a full-antagonist ligand series lacking the prototypical ligand side chain that has been universally used to engender antagonism of ERα through poorly understood structural mechanisms. A series of crystal structures and phenotypic assays reveal a structure-based design strategy with separate design elements for antagonism and degradation of the receptor, and access to a structurally distinct space for further improvements in ligand design. Understanding structural rules that guide ligands to produce diverse ERα-mediated phenotypes has broad implications for the treatment of breast cancer and other estrogen-sensitive aspects of human health including bone homeostasis, energy metabolism, and autoimmunity.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Receptores de Estrógenos/antagonistas & inhibidores , Antineoplásicos/química , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Ligandos , Modelos Moleculares , Estructura Molecular , Receptores de Estrógenos/metabolismo , Relación Estructura-Actividad
4.
Methods ; 144: 134-151, 2018 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-29678586

RESUMEN

Mass spectrometry (MS)-based methods for analyzing protein higher order structures have gained increasing application in the field of biopharmaceutical development. The predominant methods used in this area include native MS, hydrogen deuterium exchange-MS, covalent labeling, cross-linking and limited proteolysis. These MS-based methods will be briefly described in this article, followed by a discussion on how these methods contribute at different stages of discovery and development of protein therapeutics.


Asunto(s)
Medición de Intercambio de Deuterio/métodos , Desarrollo de Medicamentos/métodos , Espectrometría de Masas/métodos , Proteínas Recombinantes/metabolismo , Animales , Productos Biológicos/química , Productos Biológicos/metabolismo , Humanos , Conformación Proteica , Proteínas Recombinantes/química
5.
J Proteome Res ; 17(4): 1700-1711, 2018 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-29518331

RESUMEN

Regulator of G Protein Signaling 14 (RGS14) is a complex scaffolding protein that integrates G protein and MAPK signaling pathways. In the adult mouse brain, RGS14 is predominantly expressed in hippocampal CA2 neurons where it naturally inhibits synaptic plasticity and hippocampus-dependent learning and memory. However, the signaling proteins that RGS14 natively engages to regulate plasticity are unknown. Here, we show that RGS14 exists in a high-molecular-weight protein complex in brain. To identify RGS14 neuronal interacting partners, endogenous RGS14 immunoprecipitated from mouse brain was subjected to mass spectrometry and proteomic analysis. We find that RGS14 interacts with key postsynaptic proteins that regulate plasticity. Gene ontology analysis reveals the most enriched RGS14 interactors have functional roles in actin-binding, calmodulin(CaM)-binding, and CaM-dependent protein kinase (CaMK) activity. We validate these findings using biochemical assays that identify interactions with two previously unknown binding partners. We report that RGS14 directly interacts with Ca2+/CaM and is phosphorylated by CaMKII in vitro. Lastly, we detect that RGS14 associates with CaMKII and CaM in hippocampal CA2 neurons. Taken together, these findings demonstrate that RGS14 is a novel CaM effector and CaMKII phosphorylation substrate thereby providing new insight into mechanisms by which RGS14 controls plasticity in CA2 neurons.


Asunto(s)
Química Encefálica , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Hipocampo/química , Proteínas RGS/metabolismo , Animales , Región CA2 Hipocampal/citología , Calcio/metabolismo , Hipocampo/metabolismo , Ratones , Plasticidad Neuronal , Neuronas/metabolismo , Fosforilación , Unión Proteica , Proteómica
6.
J Biol Chem ; 292(30): 12653-12666, 2017 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-28615457

RESUMEN

AMP-activated protein kinase (AMPK) is a central cellular energy sensor that adapts metabolism and growth to the energy state of the cell. AMPK senses the ratio of adenine nucleotides (adenylate energy charge) by competitive binding of AMP, ADP, and ATP to three sites (CBS1, CBS3, and CBS4) in its γ-subunit. Because these three binding sites are functionally interconnected, it remains unclear how nucleotides bind to individual sites, which nucleotides occupy each site under physiological conditions, and how binding to one site affects binding to the other sites. Here, we comprehensively analyze nucleotide binding to wild-type and mutant AMPK protein complexes by quantitative competition assays and by hydrogen-deuterium exchange MS. We also demonstrate that NADPH, in addition to the known AMPK ligand NADH, directly and competitively binds AMPK at the AMP-sensing CBS3 site. Our findings reveal how AMP binding to one site affects the conformation and adenine nucleotide binding at the other two sites and establish CBS3, and not CBS1, as the high affinity exchangeable AMP/ADP/ATP-binding site. We further show that AMP binding at CBS4 increases AMP binding at CBS3 by 2 orders of magnitude and reverses the AMP/ATP preference of CBS3. Together, these results illustrate how the three CBS sites collaborate to enable highly sensitive detection of cellular energy states to maintain the tight ATP homeostastis required for cellular metabolism.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Adenina/metabolismo , Nucleótidos/metabolismo , Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/genética , Adenina/química , Sitios de Unión , Humanos , Modelos Moleculares , Nucleótidos/química
7.
J Biol Chem ; 291(49): 25281-25291, 2016 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-27694446

RESUMEN

Liver receptor homolog 1 (NR5A2, LRH-1) is an orphan nuclear hormone receptor that regulates diverse biological processes, including metabolism, proliferation, and the resolution of endoplasmic reticulum stress. Although preclinical and cellular studies demonstrate that LRH-1 has great potential as a therapeutic target for metabolic diseases and cancer, development of LRH-1 modulators has been difficult. Recently, systematic modifications to one of the few known chemical scaffolds capable of activating LRH-1 failed to improve efficacy substantially. Moreover, mechanisms through which LRH-1 is activated by synthetic ligands are entirely unknown. Here, we use x-ray crystallography and other structural methods to explore conformational changes and receptor-ligand interactions associated with LRH-1 activation by a set of related agonists. Unlike phospholipid LRH-1 ligands, these agonists bind deep in the pocket and do not interact with residues near the mouth nor do they expand the pocket like phospholipids. Unexpectedly, two closely related agonists with similar efficacies (GSK8470 and RJW100) exhibit completely different binding modes. The dramatic repositioning is influenced by a differential ability to establish stable face-to-face π-π-stacking with the LRH-1 residue His-390, as well as by a novel polar interaction mediated by the RJW100 hydroxyl group. The differing binding modes result in distinct mechanisms of action for the two agonists. Finally, we identify a network of conserved water molecules near the ligand-binding site that are important for activation by both agonists. This work reveals a previously unappreciated complexity associated with LRH-1 agonist development and offers insights into rational design strategies.


Asunto(s)
Compuestos de Anilina/química , Compuestos Bicíclicos con Puentes/química , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/química , Cristalografía por Rayos X , Humanos , Dominios Proteicos
8.
J Biol Chem ; 290(14): 9037-49, 2015 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-25666614

RESUMEN

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gαi/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gαi1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gαo-AlF4(-). Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gαi1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo-AlF4(-) and an AlF4(-)-insensitive mutant (G42R) of Gαi1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gαi1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Proteínas RGS/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Región CA2 Hipocampal/citología , Región CA2 Hipocampal/metabolismo , Cartilla de ADN , Células HeLa , Humanos , Neuronas/metabolismo , Ratas
9.
Anal Chem ; 88(12): 6607-14, 2016 06 21.
Artículo en Inglés | MEDLINE | ID: mdl-27224086

RESUMEN

Hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) is an information-rich biophysical method for the characterization of protein dynamics. Successful applications of differential HDX-MS include the characterization of protein-ligand binding. A single differential HDX-MS data set (protein ± ligand) is often comprised of more than 40 individual HDX-MS experiments. To eliminate laborious manual processing of samples, and to minimize random and gross errors, automated systems for HDX-MS analysis have become routine in many laboratories. However, an automated system, while less prone to random errors introduced by human operators, may have systematic errors that go unnoticed without proper detection. Although the application of automated (and manual) HDX-MS has become common, there are only a handful of studies reporting the systematic evaluation of the performance of HDX-MS experiments, and no reports have been published describing a cross-site comparison of HDX-MS experiments. Here, we describe an automated HDX-MS platform that operates with a parallel, two-trap, two-column configuration that has been installed in two remote laboratories. To understand the performance of the system both within and between laboratories, we have designed and completed a test-retest repeatability study for differential HDX-MS experiments implemented at each of two laboratories, one in Florida and the other in Spain. This study provided sufficient data to do both within and between laboratory variability assessments. Initial results revealed a systematic run-order effect within one of the two systems. Therefore, the study was repeated, and this time the conclusion was that the experimental conditions were successfully replicated with minimal systematic error.


Asunto(s)
Medición de Intercambio de Deuterio/métodos , Espectrometría de Masas/métodos , Análisis de Varianza , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Deuterio/análisis , Medición de Intercambio de Deuterio/instrumentación , Hidrógeno/análisis , Ligandos , Espectrometría de Masas/instrumentación , Péptidos/análisis , Proteínas/química , Receptores de Calcitriol/química , Reproducibilidad de los Resultados
10.
Proc Natl Acad Sci U S A ; 110(28): 11475-80, 2013 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-23798447

RESUMEN

The genetic and biophysical mechanisms by which new protein functions evolve is a central question in evolutionary biology, biochemistry, and biophysics. Of particular interest is whether major shifts in protein function are caused by a few mutations of large effect and, if they are, the mechanisms that mediate these changes. Here we combine ancestral protein reconstruction with genetic manipulation and explicit studies of protein structure and dynamics to dissect an ancient and discrete shift in ligand specificity in the steroid receptors, a family of biologically essential hormone-controlled transcription factors. We previously found that the ancestor of the entire steroid receptor family was highly specific for estrogens, but its immediate phylogenetic descendant was sensitive only to androgens, progestogens, and corticosteroids. Here we show that this shift in function was driven primarily by two historical amino acid changes, which caused a ∼70,000-fold change in the ancestral protein's specificity. These replacements subtly changed the chemistry of two amino acids, but they dramatically reduced estrogen sensitivity by introducing an excess of interaction partners into the receptor/estrogen complex, inducing a frustrated ensemble of suboptimal hydrogen bond networks unique to estrogens. This work shows how the protein's architecture and dynamics shaped its evolution, amplifying a few biochemically subtle mutations into major shifts in the energetics and function of the protein.


Asunto(s)
Biofisica , Evolución Molecular , Mutación , Receptores de Esteroides/genética , Ligandos , Modelos Moleculares , Filogenia , Unión Proteica , Receptores de Esteroides/química , Receptores de Esteroides/metabolismo
11.
J Biol Chem ; 289(21): 14941-54, 2014 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-24692551

RESUMEN

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain "activating" fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARß/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing ß2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling.


Asunto(s)
Proteínas de Unión a Ácidos Grasos/metabolismo , Ácidos Grasos/metabolismo , PPAR gamma/metabolismo , PPAR-beta/metabolismo , Animales , Ácido Araquidónico/química , Ácido Araquidónico/metabolismo , Ácido Araquidónico/farmacología , Sitios de Unión , Células COS , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Chlorocebus aethiops , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Proteínas de Unión a Ácidos Grasos/química , Proteínas de Unión a Ácidos Grasos/genética , Ácidos Grasos/química , Ácidos Grasos/farmacología , Humanos , Ligandos , Ácido Linoleico/química , Ácido Linoleico/metabolismo , Ácido Linoleico/farmacología , Células MCF-7 , Modelos Moleculares , Estructura Molecular , Mutación , Señales de Localización Nuclear/química , Señales de Localización Nuclear/metabolismo , PPAR gamma/genética , PPAR-beta/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos
12.
Anal Chem ; 87(7): 4015-4022, 2015 Apr 07.
Artículo en Inglés | MEDLINE | ID: mdl-25763479

RESUMEN

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein-ligand and protein-protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched ((15)N-labeled) p51 and unlabeled p66. To enable detection of (15)N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% (15)N model. Our results demonstrated that (15)N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but (15)N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.


Asunto(s)
Medición de Intercambio de Deuterio , Transcriptasa Inversa del VIH/análisis , Transcriptasa Inversa del VIH/química , Espectrometría de Masas , Isótopos de Nitrógeno
13.
J Biol Chem ; 288(42): 30285-30299, 2013 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-23995840

RESUMEN

The N-terminal domain (NTD) of steroid receptors harbors a transcriptional activation function (AF1) that is composed of an intrinsically disordered polypeptide. We examined the interaction of the TATA-binding protein (TBP) with the NTD of the progesterone receptor (PR) and its ability to regulate AF1 activity through coupled folding and binding. As assessed by solution phase biophysical methods, the isolated NTD of PR contains a large content of random coil, and it is capable of adopting secondary α-helical structure and more stable tertiary folding either in the presence of the natural osmolyte trimethylamine-N-oxide or through a direct interaction with TBP. Hydrogen-deuterium exchange coupled with mass spectrometry confirmed the highly dynamic intrinsically disordered property of the NTD within the context of full-length PR. Deletion mapping and point mutagenesis defined a region of the NTD (amino acids 350-428) required for structural folding in response to TBP interaction. Overexpression of TBP in cells enhanced transcriptional activity mediated by the PR NTD, and deletion mutations showed that a region (amino acids 327-428), similar to that required for TBP-induced folding, was required for functional response. TBP also increased steroid receptor co-activator 1 (SRC-1) interaction with the PR NTD and cooperated with SRC-1 to stimulate NTD-dependent transcriptional activity. These data suggest that TBP can mediate structural reorganization of the NTD to facilitate the binding of co-activators required for maximal transcriptional activation.


Asunto(s)
Coactivador 1 de Receptor Nuclear/metabolismo , Pliegue de Proteína , Receptores de Progesterona/metabolismo , Proteína de Unión a TATA-Box/metabolismo , Activación Transcripcional/fisiología , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Coactivador 1 de Receptor Nuclear/química , Coactivador 1 de Receptor Nuclear/genética , Mutación Puntual , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores de Progesterona/química , Receptores de Progesterona/genética , Eliminación de Secuencia , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética
16.
Angew Chem Int Ed Engl ; 53(1): 132-5, 2014 Jan 03.
Artículo en Inglés | MEDLINE | ID: mdl-24254636

RESUMEN

The X-ray crystal structure of a bovine antibody (BLV1H12) revealed a unique structure in its ultralong heavy chain complementarity determining region 3 (CDR3H) that folds into a solvent-exposed ß-strand "stalk" fused to a disulfide crosslinked "knob" domain. We have substituted an antiparallel heterodimeric coiled-coil motif for the ß-strand stalk in this antibody. The resulting antibody (Ab-coil) expresses in mammalian cells and has a stability similar to that of the parent bovine antibody. MS analysis of H-D exchange supports the coiled-coil structure of the substituted peptides. Substitution of the knob-domain of Ab-coil with bovine granulocyte colony-stimulating factor (bGCSF) results in a stably expressed chimeric antibody, which proliferates mouse NFS-60 cells with a potency comparable to that of bGCSF. This work demonstrates the utility of this novel coiled-coil CDR3 motif as a means for generating stable, potent antibody fusion proteins with useful pharmacological properties.


Asunto(s)
Péptidos/química , Animales , Bovinos , Proliferación Celular , Dicroismo Circular , Ratones , Modelos Moleculares , Ingeniería de Proteínas , Estructura Secundaria de Proteína
17.
J Biol Chem ; 287(53): 44546-60, 2012 Dec 28.
Artículo en Inglés | MEDLINE | ID: mdl-23132854

RESUMEN

Control of gene transcription by glucocorticoid receptors (GRs) is important for many physiological processes. Like other steroid hormone receptors, the regulation of target genes by GR is mediated by two transactivation domains: activation function 1 (AF1) in the N-terminal domain and AF2 in the C-terminal ligand-binding domain (LBD). Full receptor activity requires both AF1 and -2 plus assorted coregulatory proteins. Crystal structures of the ligand-bound LBD have provided insight regarding how AF2 interacts with specific coactivators. However, despite its being the major activation domain of GRs, knowledge of AF1 structure/function has languished. This is mainly because of the highly disorganized structure of the GR N-terminal domain. This lack of AF1 structure is shared by all members of the steroid/nuclear receptor superfamily for which it has been examined and AF1 is thought to allow productive interactions with assorted cofactors via protein-induced changes in secondary/tertiary structures. To date, there are no reports of a classical coactivator altering the secondary/tertiary structure of the GR AF1 domain. Earlier, we reported an N-terminal fragment of the p160 coactivator TIF2, called TIF2.0, that binds the GR N-terminal domain and alters GR transcriptional activity. We therefore proposed that TIF2.0 binding to AF1 changes both its conformation and transcriptional activity. We now report that TIF2.0 interacts with the GR AF1 domain to increase the amount of α-helical structure in the complex. Furthermore, TIF2 coactivator activity is observed in the absence of the GR LBD in a manner that requires the AF1 domain. This contrasts with previous models where TIF2 receptor interaction domains binding to GR LBD somehow alter AF1 conformation. Our results establish for the first time that coactivators can modify the structure of the AF1 domain directly via the binding of a second region of the coactivator and suggest a molecular explanation for how coactivators increase the transcriptional activity of GR-agonist complexes.


Asunto(s)
Coactivador 2 del Receptor Nuclear/química , Coactivador 2 del Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Cinética , Datos de Secuencia Molecular , Coactivador 2 del Receptor Nuclear/genética , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Receptores de Glucocorticoides/genética , Activación Transcripcional
18.
Biol Chem ; 393(7): 617-29, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-22944666

RESUMEN

The members of the cation diffusion facilitator (CDF) family transport heavy metal ions and play an important function in zinc ion homeostasis of the cell. A recent structure of an Escherichia coli CDF transporter protein YiiP has revealed its dimeric nature and autoregulatory zinc transport mechanism. Here, we report the cloning and heterologous production of four different CDF transporters, two each from the pathogenic mesophilic bacterium Salmonella typhimurium and from the hyperthermophilic bacterium Aquifex aeolicus, in E. coli host cells. STM0758 of S. typhimurium was able to restore resistance to zinc ions when tested by complementation assays in the zinc-sensitive GG48 strain. Furthermore, copurification of bicistronically produced STM0758 and cross-linking experiments with the purified protein have revealed its possible oligomeric nature. The interaction between heavy metal ions and Aq_2073 of A. aeolicus was investigated by titration calorimetry. The entropy-driven, high-affinity binding of two Cd2+ and two Zn2+ per protein monomer with Kd values of around 100 nm and 1 µm, respectively, was observed. In addition, at least one more Zn2+ can be bound per monomer with low affinity. This low-affinity site is likely to possess a functional role contributing to Zn2+ transport across membranes.


Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/metabolismo , Proteínas de Transporte de Catión/biosíntesis , Proteínas de Transporte de Catión/metabolismo , Bacterias Gramnegativas/genética , Salmonella typhimurium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Clonación Molecular , Biología Computacional , Escherichia coli/citología , Escherichia coli/genética , Metales Pesados/metabolismo , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Termodinámica
19.
Nat Commun ; 10(1): 94, 2019 01 09.
Artículo en Inglés | MEDLINE | ID: mdl-30626866

RESUMEN

Histone demethylase KDM5A removes methyl marks from lysine 4 of histone H3 and is often overexpressed in cancer. The in vitro demethylase activity of KDM5A is allosterically enhanced by binding of its product, unmodified H3 peptides, to its PHD1 reader domain. However, the molecular basis of this allosteric enhancement is unclear. Here we show that saturation of the PHD1 domain by the H3 N-terminal tail peptides stabilizes binding of the substrate to the catalytic domain and improves the catalytic efficiency of demethylation. When present in saturating concentrations, differently modified H3 N-terminal tail peptides have a similar effect on demethylation. However, they vary greatly in their affinity towards the PHD1 domain, suggesting that H3 modifications can tune KDM5A activity. Furthermore, hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) experiments reveal conformational changes in the allosterically enhanced state. Our findings may enable future development of anti-cancer therapies targeting regions involved in allosteric regulation.


Asunto(s)
Histonas/metabolismo , Proteína 2 de Unión a Retinoblastoma/metabolismo , Animales , Dominio Catalítico , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Proteína 2 de Unión a Retinoblastoma/genética , Células Sf9
20.
Biochemistry ; 47(49): 12964-73, 2008 Dec 09.
Artículo en Inglés | MEDLINE | ID: mdl-19006334

RESUMEN

Temperature sensitive (ts) mutants are widely used to reversibly modulate protein function in vivo and to understand functions of essential genes. Despite this, little is known about the protein structural features and mechanisms responsible for generating a ts phenotype. Also, such mutants are often difficult to isolate, limiting their use. In this study, a library consisting of 75% of all possible single-site mutants of the 101-residue, homodimeric Escherichia coli toxin CcdB was constructed. Mutants were characterized in terms of their activity at two different temperatures and at six different expression levels. Of the total of 1430 single-site mutants that were screened, 231 (16%) mutants showed a ts phenotype. The bulk of these consisted of 120 ts mutants found at all 22 buried sites and 34 ts mutants at all seven active site residues involved in binding DNA gyrase. Of the remaining ts mutants, 16 were found at residues in van der Waals contact with active site residues, 36 were at partially buried residues, and 30 resulted from introduction of Pro. Thus virtually all ts mutants could be rationalized in terms of the structure of the native protein and without knowledge of folding pathways. Data were analyzed to obtain insights into molecular features responsible for the ts phenotype and to outline structure- and sequence-based criteria for designing ts mutants of any globular protein. The criteria were validated by successful prediction of ts mutants of three other unrelated proteins, TBP, T4 lysozyme, and Gal4.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Escherichia coli/genética , Mutagénesis , Proteínas Mutantes/química , Proteínas Mutantes/genética , Temperatura , Secuencia de Aminoácidos , Arabinosa/farmacología , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Dominio Catalítico , Girasa de ADN/genética , Girasa de ADN/metabolismo , Proteínas de Unión al ADN , Dimerización , Escherichia coli/química , Datos de Secuencia Molecular , Muramidasa/química , Muramidasa/genética , Muramidasa/metabolismo , Proteínas Mutantes/metabolismo , Fenotipo , Conformación Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relación Estructura-Actividad , Proteína de Unión a TATA-Box/química , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA