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1.
Protein Sci ; 26(11): 2187-2194, 2017 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-28801928

RESUMEN

We have previously shown that monomeric globular αß-proteins can be designed de novo with considerable control over topology, size, and shape. In this paper, we investigate the design of cyclic homo-oligomers from these starting points. We experimented with both keeping the original monomer backbones fixed during the cyclic docking and design process, and allowing the backbone of the monomer to conform to that of adjacent subunits in the homo-oligomer. The latter flexible backbone protocol generated designs with shape complementarity approaching that of native homo-oligomers, but experimental characterization showed that the fixed backbone designs were more stable and less aggregation prone. Designed C2 oligomers with ß-strand backbone interactions were structurally confirmed through x-ray crystallography and small-angle X-ray scattering (SAXS). In contrast, C3-C5 designed homo-oligomers with primarily nonpolar residues at interfaces all formed a range of oligomeric states. Taken together, our results suggest that for homo-oligomers formed from globular building blocks, improved structural specificity will be better achieved using monomers with increased shape complementarity and with more polar interfaces.


Asunto(s)
Ingeniería de Proteínas , Subunidades de Proteína/química , Proteínas/química , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Electricidad Estática , Difracción de Rayos X
2.
Protein Sci ; 25(1): 30-45, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26293815

RESUMEN

We have developed an online NMR / X-ray Structure Pair Data Repository. The NIGMS Protein Structure Initiative (PSI) has provided many valuable reagents, 3D structures, and technologies for structural biology. The Northeast Structural Genomics Consortium was one of several PSI centers. NESG used both X-ray crystallography and NMR spectroscopy for protein structure determination. A key goal of the PSI was to provide experimental structures for at least one representative of each of hundreds of targeted protein domain families. In some cases, structures for identical (or nearly identical) constructs were determined by both NMR and X-ray crystallography. NMR spectroscopy and X-ray diffraction data for 41 of these "NMR / X-ray" structure pairs determined using conventional triple-resonance NMR methods with extensive sidechain resonance assignments have been organized in an online NMR / X-ray Structure Pair Data Repository. In addition, several NMR data sets for perdeuterated, methyl-protonated protein samples are included in this repository. As an example of the utility of this repository, these data were used to revisit questions about the precision and accuracy of protein NMR structures first outlined by Levy and coworkers several years ago (Andrec et al., Proteins 2007;69:449-465). These results demonstrate that the agreement between NMR and X-ray crystal structures is improved using modern methods of protein NMR spectroscopy. The NMR / X-ray Structure Pair Data Repository will provide a valuable resource for new computational NMR methods development.


Asunto(s)
Cristalografía por Rayos X , Bases de Datos de Proteínas , Resonancia Magnética Nuclear Biomolecular , Modelos Moleculares , Conformación Proteica , Proteínas/química
3.
Structure ; 23(8): 1382-1393, 2015 Aug 04.
Artículo en Inglés | MEDLINE | ID: mdl-26165597

RESUMEN

RAS binding is a critical step in the activation of BRAF protein serine/threonine kinase and stimulation of the mitogen-activated protein kinase signaling pathway. Mutations in both RAS and BRAF are associated with many human cancers. Here, we report the solution nuclear magnetic resonance (NMR) and X-ray crystal structures of the RAS-binding domain (RBD) from human BRAF. We further studied the complex between BRAF RBD and the GppNHp bound form of HRAS in solution. Backbone, side-chain, and (19)F NMR chemical shift perturbations reveal unexpected changes distal to the RAS-binding face that extend through the core of the RBD structure. Moreover, backbone amide hydrogen/deuterium exchange NMR data demonstrate conformational ensemble changes in the RBD core structure upon complex formation. These changes in BRAF RBD reveal a basis for allosteric regulation of BRAF structure and function, and suggest a mechanism by which RAS binding can signal the drastic domain rearrangements required for activation of BRAF kinase.


Asunto(s)
Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas p21(ras)/química , Regulación Alostérica , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Transducción de Señal
4.
Structure ; 22(3): 488-95, 2014 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-24440517

RESUMEN

The bacteriophage λ Q protein is a transcription antitermination factor that controls expression of the phage late genes as a stable component of the transcription elongation complex. To join the elongation complex, λQ binds a specific DNA sequence element and interacts with RNA polymerase that is paused during early elongation. λQ binds to the paused early-elongation complex through interactions between λQ and two regions of RNA polymerase: region 4 of the σ(70) subunit and the flap region of the ß subunit. We present the 2.1 Å resolution crystal structure of a portion of λQ containing determinants for interaction with DNA, interaction with region 4 of σ(70), and interaction with the ß flap. The structure provides a framework for interpreting prior genetic and biochemical analysis and sets the stage for future structural studies to elucidate the mechanism by which λQ alters the functional properties of the transcription elongation complex.


Asunto(s)
Proteínas Virales/química , Proteínas Virales/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Virales/genética , Zinc/metabolismo
5.
J Struct Funct Genomics ; 13(3): 177-83, 2012 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-22843345

RESUMEN

Recent studies of signal transduction in bacteria have revealed a unique second messenger, bis-(3'-5')-cyclic dimeric GMP (c-di-GMP), which regulates transitions between motile states and sessile states, such as biofilms. C-di-GMP is synthesized from two GTP molecules by diguanylate cyclases (DGC). The catalytic activity of DGCs depends on a conserved GG(D/E)EF domain, usually part of a larger multi-domain protein organization. The domains other than the GG(D/E)EF domain often control DGC activation. This paper presents the 1.83 Å crystal structure of an isolated catalytically competent GG(D/E)EF domain from the A1U3W3_MARAV protein from Marinobacter aquaeolei. Co-crystallization with GTP resulted in enzymatic synthesis of c-di-GMP. Comparison with previously solved DGC structures shows a similar orientation of c-di-GMP bound to an allosteric regulatory site mediating feedback inhibition of the enzyme. Biosynthesis of c-di-GMP in the crystallization reaction establishes that the enzymatic activity of this DGC domain does not require interaction with regulatory domains.


Asunto(s)
Proteínas Bacterianas/química , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/química , Marinobacter/química , Liasas de Fósforo-Oxígeno/química , Regulación Alostérica , Sitio Alostérico , Secuencia de Aminoácidos , Secuencia Conservada , Cristalografía por Rayos X/métodos , GMP Cíclico/biosíntesis , GMP Cíclico/química , Activación Enzimática , Guanosina Trifosfato/química , Marinobacter/enzimología , Datos de Secuencia Molecular , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Análisis de Secuencia de Proteína
6.
Protein Pept Lett ; 19(2): 194-7, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21933118

RESUMEN

Human retinoblastoma binding protein 9 (RBBP9) is an interacting partner of the retinoblastoma susceptibility protein (Rb). RBBP9 is a tumor-associated protein required for pancreatic neoplasia, affects cell cycle control, and is involved in the TGF-ß signalling pathway. Sequence analysis suggests that RBBP9 belongs to the α/ß hydrolase superfamily of enzymes. The serine hydrolase activity of RBBP9 is required for development of pancreatic carcinomas in part by inhibiting TGF-ß antiproliferative signaling through suppressing Smad2/3 phosphorylation. The crystal structure of human RBBP9 confirms the α/ß hydrolase fold, with a six-stranded parallel ß-sheet flanked by α helixes. The structure of RBBP9 resembles that of the YdeN protein from Bacillus subtilis, which is suggested to have carboxylesterase activity. RBBP9 contains a Ser75-His165-Asp138 catalytic triad, situated in a prominent pocket on the surface of the protein. The side chains of the LxCxE sequence motif that is important for interaction with Rb is mostly buried in the structure. Structure- function studies of RBBP9 suggest possible routes for novel cancer drug discovery programs.


Asunto(s)
Carcinoma/genética , Proteínas de Ciclo Celular/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de Neoplasias/fisiología , Neoplasias Pancreáticas/genética , Carcinoma/enzimología , Carcinoma/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Biológicos , Modelos Moleculares , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/enzimología , Neoplasias Pancreáticas/metabolismo , Conformación Proteica , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/fisiología , Relación Estructura-Actividad
7.
Nature ; 473(7348): 540-3, 2011 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-21532589

RESUMEN

Molecular replacement procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods, have allowed the rapid solution of large numbers of protein crystal structures. Despite extensive work, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modelling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modelling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction data sets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate that the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction data sets of better than 3.2 Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.


Asunto(s)
Biología Computacional/métodos , Modelos Moleculares , Proteínas/química , Homología Estructural de Proteína , Cristalografía por Rayos X , Bases de Datos de Proteínas , Electrones , Alineación de Secuencia , Homología de Secuencia de Aminoácido
9.
Protein Sci ; 16(3): 535-8, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17322535

RESUMEN

We report here the crystal structure at 2.0 A resolution of the AGR_C_4470p protein from the Gram-negative bacterium Agrobacterium tumefaciens. The protein is a tightly associated dimer, each subunit of which bears strong structural homology with the two domains of the heme utilization protein ChuS from Escherichia coli and HemS from Yersinia enterocolitica. Remarkably, the organization of the AGR_C_4470p dimer is the same as that of the two domains in ChuS and HemS, providing structural evidence that these two proteins evolved by gene duplication. However, the binding site for heme, while conserved in HemS and ChuS, is not conserved in AGR_C_4470p, suggesting that it probably has a different function. This is supported by the presence of two homologs of AGR_C_4470p in E. coli, in addition to the ChuS protein.


Asunto(s)
Agrobacterium tumefaciens/metabolismo , Proteínas Bacterianas/química , Proteínas de Transporte de Membrana/química , Oxidorreductasas/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Hemo Oxigenasa (Desciclizante)/química , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Alineación de Secuencia
10.
Proc Natl Acad Sci U S A ; 104(2): 473-8, 2007 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-17197414

RESUMEN

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-A resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.


Asunto(s)
Triptófano Oxigenasa/química , Triptófano Oxigenasa/metabolismo , Sitio Alostérico , Secuencia de Aminoácidos , Catálisis , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Indolamina-Pirrol 2,3,-Dioxigenasa/química , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Shewanella/enzimología , Shewanella/genética , Electricidad Estática , Especificidad por Sustrato , Triptófano Oxigenasa/genética , Xanthomonas campestris/enzimología , Xanthomonas campestris/genética
11.
EMBO J ; 25(19): 4458-67, 2006 Oct 04.
Artículo en Inglés | MEDLINE | ID: mdl-16977317

RESUMEN

CapG is the only member of the gelsolin family unable to sever actin filaments. Changing amino acids 84-91 (severing domain) and 124-137 (WH2-containing segment) simultaneously to the sequences of gelsolin results in a mutant, CapG-sev, capable of severing actin filaments. The gain of severing function does not alter actin filament capping, but is accompanied by a higher affinity for monomeric actin, and the capacity to bind and sequester two actin monomers. Analysis of CapG-sev crystal structure suggests a more loosely folded inactive conformation than gelsolin, with a shorter S1-S2 latch. Calcium binding to S1 opens this latch and S1 becomes separated from a closely interfaced S2-S3 complex by an extended arm consisting of amino acids 118-137. Modeling with F-actin predicts that the length of this WH2-containing arm is critical for severing function, and the addition of a single amino acid (alanine or histidine) eliminates CapG-sev severing activity, confirming this prediction. We conclude that efficient severing utilizes two actin monomer-binding sites, and that the length of the WH2-containing segment is a critical functional determinant for severing.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Gelsolina/química , Gelsolina/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Citoesqueleto de Actina/química , Actinas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Cristalografía por Rayos X , Gelsolina/genética , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Secundaria de Proteína , Conejos , Relación Estructura-Actividad
12.
J Biol Chem ; 281(11): 7533-45, 2006 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-16330546

RESUMEN

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism.


Asunto(s)
Oxo-Ácido-Liasas/química , 2-Isopropilmalato Sintasa/química , Secuencia de Aminoácidos , Ácido Aspártico/química , Bacillus subtilis/enzimología , Sitios de Unión , Brucella melitensis/enzimología , Carbono/química , Catálisis , Dominio Catalítico , Cationes , Cromatografía en Gel , Cristalografía por Rayos X , Humanos , Cinética , Luz , Lisina/química , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estrés Oxidativo , Mutación Puntual , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Dispersión de Radiación , Homología de Secuencia de Aminoácido , Estereoisomerismo
13.
J Biol Chem ; 280(48): 40328-36, 2005 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-16210326

RESUMEN

Bacillus subtilis PaiA has been implicated in the negative control of sporulation as well as production of degradative enzymes. PaiA shares recognizable sequence homology with N-acetyltransferases, including those that can acetylate spermidine/spermine substrates. We have determined the crystal structure of PaiA in complex with CoA at 1.9 A resolution and found that PaiA is a member of the N-acetyltransferase superfamily of enzymes. Unexpectedly, we observed the binding of an oxidized CoA dimer in the active site of PaiA, and the structural information suggests the substrates of the enzyme could be linear, positively charged compounds. Our biochemical characterization is also consistent with this possibility, since purified PaiA possesses N1-acetyltransferase activity toward polyamine substrates including spermidine and spermine. Further, conditional overexpression of PaiA in bacteria results in increased acetylation of endogenous spermidine pools. Thus, our structural and biochemical analyses indicate that PaiA is a novel N-acetyltransferase capable of acetylating both spermidine and spermine. In this way, the pai operon may function in regulating intracellular polyamine concentrations and/or binding capabilities. In addition to preventing toxicity due to polyamine excess, this function may also serve to regulate expression of certain bacterial gene products such as those involved in sporulation.


Asunto(s)
Acetiltransferasas/metabolismo , Acetiltransferasas/fisiología , Bacillus subtilis/metabolismo , Acetiltransferasas/química , Secuencia de Aminoácidos , Catálisis , Cationes , Cromatografía en Gel , Cristalografía por Rayos X , Dimerización , Ditiotreitol/química , Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Oxígeno/química , Poliaminas/química , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad
14.
J Biol Chem ; 277(23): 20999-1006, 2002 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-11932258

RESUMEN

An antiparallel actin dimer has been proposed to be an intermediate species during actin filament nucleation. We now show that latrunculin A, a marine natural product that inhibits actin polymerization, arrests polylysine-induced nucleation at the level of an antiparallel dimer, resulting in its accumulation. These dimers, when composed of pyrene-labeled actin subunits, give rise to a fluorescent excimer, permitting detection during polymerization in vitro. We report the crystallographic structure of the polylysine-actin-latrunculin A complex at 3.5-A resolution. The non-crystallographic contact is consistent with a dimeric structure and confirms the antiparallel orientation of its subunits. The crystallographic contacts reveal that the mobile DNase I binding loop of one subunit of a symmetry-related antiparallel actin dimer is partially stabilized in the interface between the two subunits of a second antiparallel dimer. These results provide a potential explanation for the paradoxical nucleation of actin filaments that have exclusively parallel subunits by a dimer containing antiparallel subunits.


Asunto(s)
Actinas/biosíntesis , Polilisina/fisiología , Actinas/química , Animales , Cristalografía por Rayos X , Dimerización , Modelos Moleculares , Polilisina/química , Conformación Proteica , Conejos
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