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1.
Nucleic Acids Res ; 41(4): 2673-82, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23275567

RESUMO

The serine family of site-specific DNA recombination enzymes accomplishes strand cleavage, exchange and religation using a synaptic protein tetramer. A double-strand break intermediate in which each protein subunit is covalently linked to the target DNA substrate ensures that the recombination event will not damage the DNA. The previous structure of a tetrameric synaptic complex of γδ resolvase linked to two cleaved DNA strands had suggested a rotational mechanism of recombination in which one dimer rotates 180° about the flat exchange interface for strand exchange. Here, we report the crystal structure of a synaptic tetramer of an unliganded activated mutant (M114V) of the G-segment invertase (Gin) in which one dimer half is rotated by 26° or 154° relative to the other dimer when compared with the dimers in the synaptic complex of γδ resolvase. Modeling shows that this rotational orientation of Gin is not compatible with its being able to bind uncleaved DNA, implying that this structure represents an intermediate in the process of strand exchange. Thus, our structure provides direct evidence for the proposed rotational mechanism of site-specific recombination.


Assuntos
DNA Nucleotidiltransferases/química , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Conformação Proteica , Subunidades Proteicas/química
2.
Biochim Biophys Acta ; 1804(9): 1817-31, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20580860

RESUMO

Because of their central role in programmed cell death, the caspases are attractive targets for developing new therapeutics against cancer and autoimmunity, myocardial infarction and ischemic damage, and neurodegenerative diseases. We chose to target caspase-3, an executioner caspase, and caspase-8, an initiator caspase, based on the vast amount of information linking their functions to diseases. Through a structure-based drug design approach, a number of novel beta-strand peptidomimetic compounds were synthesized. Kinetic studies of caspase-3 and caspase-8 inhibition were carried out with these urazole ring-containing irreversible peptidomimetics and a known irreversible caspase inhibitor, Z-VAD-fmk. Using a stopped-flow fluorescence assay, we were able to determine individual kinetic parameters of caspase-3 and caspase-8 inhibition by these inhibitors. Z-VAD-fmk and the peptidomimetic inhibitors inhibit caspase-3 and caspase-8 via a three-step kinetic mechanism. Inhibition of both caspase-3 and caspase-8 by Z-VAD-fmk and of caspase-3 by the peptidomimetic inhibitors proceeds via two rapid equilibrium steps followed by a relatively fast inactivation step. However, caspase-8 inhibition by the peptidomimetics goes through a rapid equilibrium step, a slow-binding reversible step, and an extremely slow inactivation step. The crystal structures of inhibitor complexes of caspases-3 and -8 validate the design of the inhibitors by illustrating in detail how they mimic peptide substrates. One of the caspase-8 structures also shows binding at a secondary, allosteric site, providing a possible route to the development of noncovalent small molecule modulators of caspase activity.


Assuntos
Caspase 3/química , Caspase 8/química , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Caspase , Cristalização , Cristalografia por Raios X , Inibidores de Cisteína Proteinase/síntese química , Humanos , Cinética , Estrutura Molecular , Conformação Proteica
3.
Bioorg Med Chem Lett ; 21(21): 6545-53, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21924613

RESUMO

Fatty acid amide hydrolase (FAAH) is an integral membrane serine hydrolase responsible for the degradation of fatty acid amide signaling molecules such as endocannabinoid anandamide (AEA), which has been shown to possess cannabinoid-like analgesic properties. Herein we report the optimization of spirocyclic 7-azaspiro[3.5]nonane and 1-oxa-8-azaspiro[4.5]decane urea covalent inhibitors of FAAH. Using an iterative design and optimization strategy, lead compounds were identified with a remarkable reduction in molecular weight and favorable CNS drug like properties. 3,4-Dimethylisoxazole and 1-methyltetrazole were identified as superior urea moieties for this inhibitor class. A dual purpose in vivo efficacy and pharmacokinetic screen was designed to be the key decision enabling experiment affording the ability to move quickly from compound synthesis to selection of preclinical candidates. On the basis of the remarkable potency, selectivity, pharmacokinetic properties and in vivo efficacy, PF-04862853 (15p) was advanced as a clinical candidate.


Assuntos
Amidoidrolases/antagonistas & inibidores , Analgésicos/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Dor/tratamento farmacológico , Compostos de Espiro/farmacologia , Administração Oral , Analgésicos/administração & dosagem , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Compostos Aza/administração & dosagem , Compostos Aza/química , Compostos Aza/farmacologia , Compostos Aza/uso terapêutico , Disponibilidade Biológica , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Inibidores Enzimáticos/uso terapêutico , Ratos , Compostos de Espiro/administração & dosagem , Compostos de Espiro/química , Compostos de Espiro/uso terapêutico
4.
Proc Natl Acad Sci U S A ; 105(35): 12820-4, 2008 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-18753625

RESUMO

The integral membrane enzyme fatty acid amide hydrolase (FAAH) hydrolyzes the endocannabinoid anandamide and related amidated signaling lipids. Genetic or pharmacological inactivation of FAAH produces analgesic, anxiolytic, and antiinflammatory phenotypes but not the undesirable side effects of direct cannabinoid receptor agonists, indicating that FAAH may be a promising therapeutic target. Structure-based inhibitor design has, however, been hampered by difficulties in expressing the human FAAH enzyme. Here, we address this problem by interconverting the active sites of rat and human FAAH using site-directed mutagenesis. The resulting humanized rat (h/r) FAAH protein exhibits the inhibitor sensitivity profiles of human FAAH but maintains the high-expression yield of the rat enzyme. We report a 2.75-A crystal structure of h/rFAAH complexed with an inhibitor, N-phenyl-4-(quinolin-3-ylmethyl)piperidine-1-carboxamide (PF-750), that shows strong preference for human FAAH. This structure offers compelling insights to explain the species selectivity of FAAH inhibitors, which should guide future drug design programs.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/química , Desenho de Fármacos , Inibidores Enzimáticos/química , Anilidas/química , Animais , Sítios de Ligação , Catálise/efeitos dos fármacos , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Humanos , Cinética , Modelos Moleculares , Piperidinas/química , Engenharia de Proteínas , Estrutura Secundária de Proteína , Ratos , Especificidade da Espécie
6.
Artigo em Inglês | MEDLINE | ID: mdl-20823544

RESUMO

Rat autotaxin has been cloned, expressed, purified to homogeneity and crystallized via hanging-drop vapour diffusion using PEG 3350 as precipitant and ammonium iodide and sodium thiocyanate as salts. The crystals diffracted to a maximum resolution of 2.05 A and belonged to space group P1, with unit-cell parameters a=53.8, b=63.3, c=70.5 A, alpha=98.8, beta=106.2, gamma=99.8 degrees. Preliminary X-ray diffraction analysis indicated the presence of one molecule per asymmetric unit, with a solvent content of 47%.


Assuntos
Diester Fosfórico Hidrolases/química , Pirofosfatases/química , Animais , Cristalização , Cristalografia por Raios X , Ratos
7.
J Med Chem ; 60(13): 5521-5542, 2017 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-28498658

RESUMO

Through fragment-based drug design focused on engaging the active site of IRAK4 and leveraging three-dimensional topology in a ligand-efficient manner, a micromolar hit identified from a screen of a Pfizer fragment library was optimized to afford IRAK4 inhibitors with nanomolar potency in cellular assays. The medicinal chemistry effort featured the judicious placement of lipophilicity, informed by co-crystal structures with IRAK4 and optimization of ADME properties to deliver clinical candidate PF-06650833 (compound 40). This compound displays a 5-unit increase in lipophilic efficiency from the fragment hit, excellent kinase selectivity, and pharmacokinetic properties suitable for oral administration.


Assuntos
Descoberta de Drogas , Quinases Associadas a Receptores de Interleucina-1/antagonistas & inibidores , Isoquinolinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Administração Oral , Relação Dose-Resposta a Droga , Humanos , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Isoquinolinas/administração & dosagem , Isoquinolinas/química , Lactamas , Modelos Moleculares , Estrutura Molecular , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade
8.
FEBS Lett ; 579(27): 6017-22, 2005 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-16226256

RESUMO

Archaeal prolyl-tRNA synthetases differ from their bacterial counterparts: they contain an additional domain (about 70 amino acids) appended to the carboxy-terminus and lack an editing domain inserted into the class II catalytic core. Biochemical and structural approaches have generated a wealth of information on amino acid and tRNA specificities for both types of ProRSs, but have left a number of aspects unexplored. We report here that the carboxy-terminal domain of Methanocaldococcus jannaschii ProRS is not involved in tRNA binding since its deletion only mildly affects the kinetic parameters for the enzyme. We also demonstrate that M. jannaschii ProRS is a homodimeric enzyme that is functionally asymmetric; only one of the two active sites at a time is able to form prolyl-adenylate, and only one tRNA molecule binds per dimer. Together with previous reports our results show that asymmetry might be a general feature of the aminoacylation reaction catalyzed by dimeric aminoacyl-tRNA synthetases from both classes.


Assuntos
Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Methanococcales/enzimologia , Aminoacil-RNA de Transferência/metabolismo , Aminoacil-tRNA Sintetases/genética , Proteínas Arqueais/genética , Sítios de Ligação , Dimerização , Methanococcales/genética , Nucleotídeos/metabolismo , Estrutura Terciária de Proteína , Deleção de Sequência , Aminoacilação de RNA de Transferência
9.
Nat Struct Mol Biol ; 18(2): 198-204, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21240271

RESUMO

Autotaxin (ATX, also known as ectonucleotide pyrophosphatase/phosphodiesterase-2, ENPP2) is a secreted lysophospholipase D that generates the lipid mediator lysophosphatidic acid (LPA), a mitogen and chemoattractant for many cell types. ATX-LPA signaling is involved in various pathologies including tumor progression and inflammation. However, the molecular basis of substrate recognition and catalysis by ATX and the mechanism by which it interacts with target cells are unclear. Here, we present the crystal structure of ATX, alone and in complex with a small-molecule inhibitor. We have identified a hydrophobic lipid-binding pocket and mapped key residues for catalysis and selection between nucleotide and phospholipid substrates. We have shown that ATX interacts with cell-surface integrins through its N-terminal somatomedin B-like domains, using an atypical mechanism. Our results define determinants of substrate discrimination by the ENPP family, suggest how ATX promotes localized LPA signaling and suggest new approaches for targeting ATX with small-molecule therapeutic agents.


Assuntos
Integrinas/metabolismo , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Pirofosfatases/química , Pirofosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Domínio Catalítico , Linhagem Celular , Cristalografia por Raios X , Humanos , Lisofosfolipídeos/metabolismo , Dados de Sequência Molecular , Mutação , Diester Fosfórico Hidrolases/genética , Ligação Proteica , Estrutura Terciária de Proteína , Pirofosfatases/genética , Ratos , Especificidade por Substrato
10.
J Mol Biol ; 400(4): 743-54, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20493882

RESUMO

The endocannabinoid system regulates a wide range of physiological processes including pain, inflammation, and cognitive/emotional states. URB597 is one of the best characterized covalent inhibitors of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH). Here, we report the structure of the FAAH-URB597 complex at 2.3 A resolution. The structure provides insights into mechanistic details of enzyme inactivation and experimental evidence of a previously uncharacterized active site water molecule that likely is involved in substrate deacylation. This water molecule is part of an extensive hydrogen-bonding network and is coordinated indirectly to residues lining the cytosolic port of the enzyme. In order to corroborate our hypothesis concerning the role of this water molecule in FAAH's catalytic mechanism, we determined the structure of FAAH conjugated to a urea-based inhibitor, PF-3845, to a higher resolution (2.4 A) than previously reported. The higher-resolution structure confirms the presence of the water molecule in a virtually identical location in the active site. Examination of the structures of serine hydrolases that are non-homologous to FAAH, such as elastase, trypsin, or chymotrypsin, shows a similarly positioned hydrolytic water molecule and suggests a functional convergence between the amidase signature enzymes and serine proteases.


Assuntos
Amidoidrolases/química , Benzamidas/química , Carbamatos/química , Inibidores Enzimáticos/química , Amidoidrolases/antagonistas & inibidores , Benzamidas/metabolismo , Carbamatos/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Inibidores Enzimáticos/metabolismo , Modelos Moleculares , Ligação Proteica , Estrutura Terciária de Proteína , Água
11.
Nat Struct Mol Biol ; 16(5): 499-508, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19396172

RESUMO

The products of recombination-activating genes RAG1 and RAG2 mediate the assembly of antigen receptor genes during lymphocyte development in a process known as V(D)J recombination. Lack of structural information for the RAG proteins has hindered mechanistic studies of this reaction. We report here the crystal structure of an essential DNA binding domain of the RAG1 catalytic core bound to its nonamer DNA recognition motif. The RAG1 nonamer binding domain (NBD) forms a tightly interwoven dimer that binds and synapses two nonamer elements, with each NBD making contact with both DNA molecules. Biochemical and biophysical experiments confirm that the two nonamers are in close proximity in the RAG1/2-DNA synaptic complex and demonstrate the functional importance of the protein-DNA contacts revealed in the structure. These findings reveal a previously unsuspected function for the NBD in DNA synapsis and have implications for the regulation of DNA binding and cleavage by RAG1 and RAG2.


Assuntos
Pareamento Cromossômico , DNA/química , Proteínas de Homeodomínio/química , Multimerização Proteica , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência de Bases , Cristalografia por Raios X , DNA/genética , Transferência Ressonante de Energia de Fluorescência , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Soluções , Eletricidade Estática
12.
Chem Biol ; 16(4): 411-20, 2009 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-19389627

RESUMO

Endocannabinoids are lipid signaling molecules that regulate a wide range of mammalian behaviors, including pain, inflammation, and cognitive/emotional state. The endocannabinoid anandamide is principally degraded by the integral membrane enzyme fatty acid amide hydrolase (FAAH), and there is currently much interest in developing FAAH inhibitors to augment endocannabinoid signaling in vivo. Here, we report the discovery and detailed characterization of a highly efficacious and selective FAAH inhibitor, PF-3845. Mechanistic and structural studies confirm that PF-3845 is a covalent inhibitor that carbamylates FAAH's serine nucleophile. PF-3845 selectively inhibits FAAH in vivo, as determined by activity-based protein profiling; raises brain anandamide levels for up to 24 hr; and produces significant cannabinoid receptor-dependent reductions in inflammatory pain. These data thus designate PF-3845 as a valuable pharmacological tool for in vivo characterization of the endocannabinoid system.


Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Dor/tratamento farmacológico , Amidoidrolases/química , Animais , Ácidos Araquidônicos/metabolismo , Encéfalo/metabolismo , Cristalografia por Raios X , Endocanabinoides , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Masculino , Dor/induzido quimicamente , Dor/imunologia , Piperazina , Piperazinas/síntese química , Piperazinas/química , Piperazinas/metabolismo , Piperidinas/síntese química , Piperidinas/química , Piperidinas/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Canabinoides/metabolismo , Relação Estrutura-Atividade , Ureia/síntese química , Ureia/química , Ureia/metabolismo
13.
Proc Natl Acad Sci U S A ; 104(8): 2620-5, 2007 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-17301225

RESUMO

A number of archaeal organisms generate Cys-tRNA(Cys) in a two-step pathway, first charging phosphoserine (Sep) onto tRNA(Cys) and subsequently converting it to Cys-tRNA(Cys). We have determined, at 3.2-A resolution, the structure of the Methanococcus maripaludis phosphoseryl-tRNA synthetase (SepRS), which catalyzes the first step of this pathway. The structure shows that SepRS is a class II, alpha(4) synthetase whose quaternary structure arrangement of subunits closely resembles that of the heterotetrameric (alphabeta)(2) phenylalanyl-tRNA synthetase (PheRS). Homology modeling of a tRNA complex indicates that, in contrast to PheRS, a single monomer in the SepRS tetramer may recognize both the acceptor terminus and anticodon of a tRNA substrate. Using a complex with tungstate as a marker for the position of the phosphate moiety of Sep, we suggest that SepRS and PheRS bind their respective amino acid substrates in dissimilar orientations by using different residues.


Assuntos
Aminoacil-tRNA Sintetases/química , Mathanococcus/química , Mathanococcus/enzimologia , Fosfosserina/metabolismo , RNA de Transferência de Cisteína/biossíntese , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Difosfatos/metabolismo , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Thermus thermophilus/enzimologia
14.
EMBO J ; 26(14): 3494-505, 2007 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-17611604

RESUMO

Replicative DNA polymerases (DNAPs) move along template DNA in a processive manner. The structural basis of the mechanism of translocation has been better studied in the A-family of polymerases than in the B-family of replicative polymerases. To address this issue, we have determined the X-ray crystal structures of phi29 DNAP, a member of the protein-primed subgroup of the B-family of polymerases, complexed with primer-template DNA in the presence or absence of the incoming nucleoside triphosphate, the pre- and post-translocated states, respectively. Comparison of these structures reveals a mechanism of translocation that appears to be facilitated by the coordinated movement of two conserved tyrosine residues into the insertion site. This differs from the mechanism employed by the A-family polymerases, in which a conserved tyrosine moves into the templating and insertion sites during the translocation step. Polymerases from the two families also interact with downstream single-stranded template DNA in very different ways.


Assuntos
Fagos Bacilares/enzimologia , DNA Polimerase Dirigida por DNA/química , Motivos de Aminoácidos , Cristalografia por Raios X , DNA Viral/metabolismo , Exonucleases/metabolismo , Modelos Moleculares , Transporte Proteico , Especificidade por Substrato , Moldes Genéticos , Água/metabolismo
15.
EMBO J ; 25(6): 1335-43, 2006 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-16511564

RESUMO

The absolute requirement for primers in the initiation of DNA synthesis poses a problem for replicating the ends of linear chromosomes. The DNA polymerase of bacteriophage phi29 solves this problem by using a serine hydroxyl of terminal protein to prime replication. The 3.0 A resolution structure shows one domain of terminal protein making no interactions, a second binding the polymerase and a third domain containing the priming serine occupying the same binding cleft in the polymerase as duplex DNA does during elongation. Thus, the progressively elongating DNA duplex product must displace this priming domain. Further, this heterodimer of polymerase and terminal protein cannot accommodate upstream template DNA, thereby explaining its specificity for initiating DNA synthesis only at the ends of the bacteriophage genome. We propose a model for the transition from the initiation to the elongation phases in which the priming domain of terminal protein moves out of the active site as polymerase elongates the primer strand. The model indicates that terminal protein should dissociate from polymerase after the incorporation of approximately six nucleotides.


Assuntos
Fagos Bacilares/enzimologia , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , Modelos Moleculares , Elongação Traducional da Cadeia Peptídica , Sítios de Ligação , Cristalografia por Raios X , Primers do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação/genética , Transcrição Gênica
16.
Proc Natl Acad Sci U S A ; 103(28): 10642-7, 2006 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-16807292

RESUMO

The structures of two mutants of the site-specific recombinase, gammadelta resolvase, that form activated tetramers have been determined. One, at 3.5-A resolution, forms a synaptic intermediate of resolvase that is covalently linked to two cleaved DNAs, whereas the other is of an unliganded structure determined at 2.1-A resolution. Comparisons of the four known tetrameric resolvase structures show that the subunits interact through the formation of a common core of four helices. The N-terminal halves of these helices superimpose well on each other, whereas the orientations of their C termini are more variable. The catalytic domains of resolvase in the unliganded structure are arranged asymmetrically, demonstrating that their positions can move substantially while preserving the four-helix core that forms the tetramer. These results suggest that the precleavage synaptic tetramer of gammadelta resolvase, whose structure is not known, may be formed by a similar four-helix core, but differ in the relative orientations of its catalytic and DNA-binding domains.


Assuntos
Recombinação Genética/fisiologia , Transposon Resolvases/química , Transposon Resolvases/fisiologia , Cristalografia por Raios X , DNA/metabolismo , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/genética , DNA Nucleotidiltransferases/fisiologia , Humanos , Proteínas Mutantes Quiméricas/química , Proteínas Mutantes Quiméricas/fisiologia , Transposon Resolvases/genética
17.
Acta Crystallogr D Biol Crystallogr ; 61(Pt 1): 67-74, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15608377

RESUMO

In 1954, Howells and colleagues described an unusual diffraction pattern from imidazole methemoglobin crystals caused by lattice-translocation defects. In these crystals, two identical lattices coexist as a single coherent mosaic block, but are translated by a fixed vector with respect to each other. The observed structure is a weighted sum of the two identical but translated structures, one from each lattice; the observed structure factors are a weighted vector sum of the two structure factors with identical unit amplitudes but shifted phases. A general procedure is described to obtain the unit amplitudes of observed structure factors from a realigned single lattice through an X-ray intensity correction. An application of this procedure is made to determine the crystal structure of phi29 DNA polymerase at 2.2 A resolution using multiple isomorphous replacement and multiwavelength anomalous dispersion methods.


Assuntos
Fagos Bacilares/enzimologia , Cristalografia por Raios X/métodos , DNA Polimerase Dirigida por DNA/química , Cristalização , Substâncias Macromoleculares , Modelos Moleculares , Modelos Estatísticos , Conformação Molecular , Conformação Proteica , Software , Difração de Raios X , Raios X
18.
Science ; 309(5738): 1210-5, 2005 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-15994378

RESUMO

The structure of a synaptic intermediate of the site-specific recombinase gammadelta resolvase covalently linked through Ser10 to two cleaved duplex DNAs has been determined at 3.4 angstrom resolution. This resolvase, activated for recombination by mutations, forms a tetramer whose structure is substantially changed from that of a presynaptic complex between dimeric resolvase and the cleavage site DNA. Because the two cleaved DNA duplexes that are to be recombined lie on opposite sides of the core tetramer, large movements of both protein and DNA are required to achieve strand exchange. The two dimers linked to the DNAs that are to be recombined are held together by a flat interface. This may allow a 180 degrees rotation of one dimer relative to the other in order to reposition the DNA duplexes for strand exchange.


Assuntos
DNA/química , DNA/metabolismo , Transposon Resolvases/química , Substituição de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Simulação por Computador , Cristalografia por Raios X , Dimerização , Modelos Moleculares , Mutação , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Recombinação Genética , Transposon Resolvases/genética , Transposon Resolvases/metabolismo
19.
Proc Natl Acad Sci U S A ; 102(18): 6407-12, 2005 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-15845765

RESUMO

Recent crystallographic studies of phi29 DNA polymerase have provided structural insights into its strand displacement and processivity. A specific insertion named terminal protein region 2 (TPR2), present only in protein-primed DNA polymerases, together with the exonuclease, thumb, and palm subdomains, forms two tori capable of interacting with DNA. To analyze the functional role of this insertion, we constructed a phi29 DNA polymerase deletion mutant lacking TPR2 amino acid residues Asp-398 to Glu-420. Biochemical analysis of the mutant DNA polymerase indicates that its DNA-binding capacity is diminished, drastically decreasing its processivity. In addition, removal of the TPR2 insertion abolishes the intrinsic capacity of phi29 DNA polymerase to perform strand displacement coupled to DNA synthesis. Therefore, the biochemical results described here directly demonstrate that TPR2 plays a critical role in strand displacement and processivity.


Assuntos
Bacteriófago T4/genética , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Modelos Moleculares , Sequência de Aminoácidos , Primers do DNA , DNA Polimerase Dirigida por DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Exodesoxirribonucleases/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Moldes Genéticos
20.
Proc Natl Acad Sci U S A ; 100(4): 1673-8, 2003 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-12578991

RESUMO

Cysteinyl-tRNA synthetase is an essential enzyme required for protein synthesis. Genes encoding this protein have not been identified in Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, or Methanopyrus kandleri. It has previously been proposed that the prolyl-tRNA synthetase (ProRS) enzymes in these organisms recognize either proline or cysteine and can aminoacylate their cognate tRNAs through a dual-specificity mechanism. We report five crystal structures at resolutions between 2.6 and 3.2 A: apo M. jannaschii ProRS, and M. thermautotrophicus ProRS in apo form and in complex with cysteinyl-sulfamoyl-, prolyl-sulfamoyl-, and alanyl-sulfamoyl-adenylates. These aminoacyl-adenylate analogues bind to a single active-site pocket and induce an identical set of conformational changes in loops around the active site when compared with the ligand-free conformation of ProRS. The cysteinyl- and prolyl-adenylate analogues have similar, nanomolar affinities for M. thermautotrophicus ProRS. Homology modeling of tRNA onto these adenylate complexes places the 3'-OH of A76 in an appropriate position for the transfer of any of the three amino acids to tRNA. Thus, these structures explain recent biochemical experiments showing that M. jannaschii ProRS misacylates tRNA(Pro) with cysteine, and argue against the proposal that these archaeal ProRS enzymes possess the dual capacity to aminoacylate both tRNA(Pro) and tRNA(Cys) with their cognate amino acids.


Assuntos
Aminoacil-tRNA Sintetases/metabolismo , Cisteína/metabolismo , RNA de Transferência de Prolina/metabolismo , Acilação , Aminoacil-tRNA Sintetases/química , Aminoacil-tRNA Sintetases/isolamento & purificação , Euryarchaeota/enzimologia , Modelos Moleculares , Ligação Proteica , Conformação Proteica
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