Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 19 de 19
Filtrar
1.
Nat Commun ; 15(1): 3544, 2024 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-38740791

RESUMO

G-protein-coupled receptors (GPCRs) play pivotal roles in various physiological processes. These receptors are activated to different extents by diverse orthosteric ligands and allosteric modulators. However, the mechanisms underlying these variations in signaling activity by allosteric modulators remain largely elusive. Here, we determine the three-dimensional structure of the µ-opioid receptor (MOR), a class A GPCR, in complex with the Gi protein and an allosteric modulator, BMS-986122, using cryogenic electron microscopy. Our results reveal that BMS-986122 binding induces changes in the map densities corresponding to R1673.50 and Y2545.58, key residues in the structural motifs conserved among class A GPCRs. Nuclear magnetic resonance analyses of MOR in the absence of the Gi protein reveal that BMS-986122 binding enhances the formation of the interaction between R1673.50 and Y2545.58, thus stabilizing the fully-activated conformation, where the intracellular half of TM6 is outward-shifted to allow for interaction with the Gi protein. These findings illuminate that allosteric modulators like BMS-986122 can potentiate receptor activation through alterations in the conformational dynamics in the core region of GPCRs. Together, our results demonstrate the regulatory mechanisms of GPCRs, providing insights into the rational development of therapeutics targeting GPCRs.


Assuntos
Microscopia Crioeletrônica , Receptores Opioides mu , Receptores Opioides mu/metabolismo , Receptores Opioides mu/química , Receptores Opioides mu/genética , Regulação Alostérica , Humanos , Ligação Proteica , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Células HEK293 , Ligantes , Modelos Moleculares , Conformação Proteica
2.
J Virol ; 98(5): e0019724, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38593321

RESUMO

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Norovirus , Humanos , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Sítios de Ligação , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/química , Proteínas do Capsídeo/metabolismo , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X , Modelos Moleculares , Norovirus/imunologia
3.
Nat Commun ; 14(1): 4103, 2023 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-37460559

RESUMO

Histone acetylation is important for the activation of gene transcription but little is known about its direct read/write mechanisms. Here, we report cryogenic electron microscopy structures in which a p300/CREB-binding protein (CBP) multidomain monomer recognizes histone H4 N-terminal tail (NT) acetylation (ac) in a nucleosome and acetylates non-H4 histone NTs within the same nucleosome. p300/CBP not only recognized H4NTac via the bromodomain pocket responsible for reading, but also interacted with the DNA minor grooves via the outside of that pocket. This directed the catalytic center of p300/CBP to one of the non-H4 histone NTs. The primary target that p300 writes by reading H4NTac was H2BNT, and H2BNTac promoted H2A-H2B dissociation from the nucleosome. We propose a model in which p300/CBP replicates histone N-terminal tail acetylation within the H3-H4 tetramer to inherit epigenetic storage, and transcribes it from the H3-H4 tetramer to the H2B-H2A dimers to activate context-dependent gene transcription through local nucleosome destabilization.


Assuntos
Histonas , Nucleossomos , Histonas/metabolismo , Proteína de Ligação a CREB/genética , Acetilação , Epigênese Genética , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
4.
iScience ; 26(6): 106955, 2023 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-37288342

RESUMO

Several antibody therapeutics have been developed against SARS-CoV-2; however, they have attenuated neutralizing ability against variants. In this study, we generated multiple broadly neutralizing antibodies from B cells of convalescents, by using two types of receptor-binding domains, Wuhan strain and the Gamma variant as bait. From 172 antibodies generated, six antibodies neutralized all strains prior to the Omicron variant, and the five antibodies were able to neutralize some of the Omicron sub-strains. Structural analysis showed that these antibodies have a variety of characteristic binding modes, such as ACE2 mimicry. We subjected a representative antibody to the hamster infection model after introduction of the N297A modification, and observed a dose-dependent reduction of the lung viral titer, even at a dose of 2 mg/kg. These results demonstrated that our antibodies have certain antiviral activity as therapeutics, and highlighted the importance of initial cell-screening strategy for the efficient development of therapeutic antibodies.

5.
iScience ; 25(12): 105596, 2022 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-36406861

RESUMO

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from patients with COVID-19-convalescent, and identified antibodies that exhibited the comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced. Our antibodies showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

6.
Nature ; 606(7916): 1027-1031, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35580630

RESUMO

Around 250 million people are infected with hepatitis B virus (HBV) worldwide1, and 15 million may also carry the satellite virus hepatitis D virus (HDV), which confers even greater risk of severe liver disease2. The HBV receptor has been identified as sodium taurocholate co-transporting polypeptide (NTCP), which interacts directly with the first 48 amino acid residues of the N-myristoylated N-terminal preS1 domain of the viral large protein3. Despite the pressing need for therapeutic agents to counter HBV, the structure of NTCP remains unsolved. This 349-residue protein is closely related to human apical sodium-dependent bile acid transporter (ASBT), another member of the solute carrier family SLC10. Crystal structures have been reported of similar bile acid transporters from bacteria4,5, and these models are believed to resemble closely both NTCP and ASBT. Here we have used cryo-electron microscopy to solve the structure of NTCP bound to an antibody, clearly showing that the transporter has no equivalent of the first transmembrane helix found in other SLC10 proteins, and that the N terminus is exposed on the extracellular face. Comparison of our structure with those of related proteins indicates a common mechanism of bile acid transport, but the NTCP structure displays an additional pocket formed by residues that are known to interact with preS1, presenting new opportunities for structure-based drug design.


Assuntos
Ácidos e Sais Biliares , Microscopia Crioeletrônica , Vírus da Hepatite B , Transportadores de Ânions Orgânicos Dependentes de Sódio , Receptores Virais , Simportadores , Anticorpos , Ácidos e Sais Biliares/metabolismo , Vírus da Hepatite B/metabolismo , Hepatócitos/metabolismo , Humanos , Transportadores de Ânions Orgânicos Dependentes de Sódio/química , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Transportadores de Ânions Orgânicos Dependentes de Sódio/ultraestrutura , Receptores Virais/química , Receptores Virais/metabolismo , Receptores Virais/ultraestrutura , Simportadores/química , Simportadores/metabolismo , Simportadores/ultraestrutura
7.
FEBS J ; 289(12): 3505-3520, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35030303

RESUMO

Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane ß-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component α-hemolysin (α-HL) and two-component γ-hemolysin (γ-HL). The α-HL forms a homo-heptamer, whereas γ-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a ß-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the ß-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of α-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT ß-barrels for use in further promising applications.


Assuntos
Toxinas Bacterianas , Proteínas Hemolisinas , Toxinas Bacterianas/metabolismo , Proteínas Hemolisinas/metabolismo , Hemólise , Leucocidinas/química , Leucocidinas/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo
8.
Sci Adv ; 7(30)2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34290093

RESUMO

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) promotes cell motility, phagocytosis, and cancer metastasis through activation of Rho guanosine triphosphatases. Engulfment and cell motility (ELMO) proteins are binding partners of DOCK and regulate Rac activation. Here, we report the cryo-electron microscopy structure of the active ELMO1-DOCK5 complex bound to Rac1 at 3.8-Å resolution. The C-terminal region of ELMO1, including the pleckstrin homology (PH) domain, aids in the binding of the catalytic DOCK homology region 2 (DHR-2) domain of DOCK5 to Rac1 in its nucleotide-free state. A complex α-helical scaffold between ELMO1 and DOCK5 stabilizes the binding of Rac1. Mutagenesis studies revealed that the PH domain of ELMO1 enhances the GEF activity of DOCK5 through specific interactions with Rac1. The structure provides insights into how ELMO modulates the biochemical activity of DOCK and how Rac selectivity is achieved by ELMO.

9.
Sci Rep ; 11(1): 5790, 2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33707468

RESUMO

Antibodies have been widely used for cancer therapy owing to their ability to distinguish cancer cells by recognizing cancer-specific antigens. Epidermal growth factor receptor (EGFR) is a promising target for the cancer therapeutics, against which several antibody clones have been developed and brought into therapeutic use. Another antibody clone, 528, is an antagonistic anti-EGFR antibody, which has been the focus of our antibody engineering studies to develop cancer drugs. In this study, we explored the interaction of 528 with the extracellular region of EGFR (sEGFR) via binding analyses and structural studies. Dot blotting experiments with heat treated sEGFR and surface plasmon resonance binding experiments revealed that 528 recognizes the tertiary structure of sEGFR and exhibits competitive binding to sEGFR with EGF and cetuximab. Single particle analysis of the sEGFR-528 Fab complex via electron microscopy clearly showed the binding of 528 to domain III of sEGFR, the domain to which EGF and cetuximab bind, explaining its antagonistic activity. Comparison between the two-dimensional class average and the cetuximab/sEGFR crystal structure revealed that 528 binds to a site that is shifted from, rather than identical to, the cetuximab epitope, and may exclude known drug-resistant EGFR mutations.


Assuntos
Cetuximab/metabolismo , Epitopos/metabolismo , Receptores ErbB/química , Receptores ErbB/metabolismo , Animais , Ligação Competitiva , Células CHO , Cetuximab/química , Cetuximab/ultraestrutura , Cricetulus , Fator de Crescimento Epidérmico/metabolismo , Epitopos/química , Receptores ErbB/ultraestrutura , Temperatura Alta , Modelos Moleculares , Ligação Proteica , Domínios Proteicos
10.
J Cell Biol ; 217(12): 4155-4163, 2018 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-30275105

RESUMO

The Tau family microtubule-associated proteins (MAPs) promote microtubule stabilization and regulate microtubule-based motility. They share the C-terminal microtubule-binding domain, which includes three to five tubulin-binding repeats. Different numbers of repeats formed by alternative splicing have distinct effects on the activities of these proteins, and the distribution of these variants regulates fundamental physiological phenomena in cells. In this study, using cryo-EM, we visualized the MAP4 microtubule complex with the molecular motor kinesin-1. MAP4 bound to the C-terminal domains of tubulins along the protofilaments stabilizes the longitudinal contacts of the microtubule. The strongest bond of MAP4 was found around the intertubulin-dimer interface such that MAP4 coexists on the microtubule with kinesin-1 bound to the intratubulin-dimer interface as well. MAP4, consisting of five repeats, further folds and accumulates above the intertubulin-dimer interface, interfering with kinesin-1 movement. Therefore, these cryo-EM studies reveal new insight into the structural basis of microtubule stabilization and inhibition of kinesin motility by the Tau family MAPs.


Assuntos
Cinesinas , Proteínas Associadas aos Microtúbulos , Microtúbulos , Humanos , Cinesinas/química , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura
11.
J Mol Biol ; 426(17): 3016-27, 2014 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-25020231

RESUMO

The major allergen, Cry j 1, was isolated from Japanese cedar Cryptomeria japonica (Cry j) pollen and was shown to react with immunoglobulin E antibodies in the sera from pollinosis patients. We previously reported that the frequency of HLA-DP5 was significantly higher in pollinosis patients and the immunodominant peptides from Cry j 1 bound to HLA-DP5 to activate Th2 cells. In the present study, we determined the crystal structure of the HLA-DP5 heterodimer in complex with a Cry j 1-derived nine-residue peptide, at 2.4Å resolution. The peptide-binding groove recognizes the minimal peptide with 10 hydrogen bonds, including those between the negatively charged P1 pocket and the Lys side chain at the first position in the peptide sequence. We confirmed that HLA-DP5 exhibits the same Cry j 1-binding mode in solution, through pull-down experiments using structure-based mutations of Cry j 1. We also identified the characteristic residues of HLA-DP5 that are responsible for the distinct properties of the groove, by comparing the structure of HLA-DP5 and the previously reported structures of HLA-DP2 in complexes with pDRA of the self-antigen. The comparison revealed that the HLA-DP5·pCry j 1 complex forms several hydrogen bond/salt bridge networks between the receptor and the antigen that were not observed in the HLA-DP2·pDRA complex. Evolutionary considerations have led us to conclude that HLA-DP5 and HLA-DP2 represent two major groups of the HLA-DP family, in which the properties of the P1 and P4 pockets have evolved and acquired the present ranges of epitope peptide-binding specificities.


Assuntos
Antígenos de Plantas/química , Cadeias alfa de HLA-DP/química , Cadeias beta de HLA-DP/química , Fragmentos de Peptídeos/química , Proteínas de Plantas/química , Animais , Sítios de Ligação , Cryptomeria/química , Cristalografia por Raios X , Cadeias alfa de HLA-DP/genética , Cadeias beta de HLA-DP/genética , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Filogenia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Células Sf9 , Spodoptera
12.
J Struct Biol ; 168(3): 575-81, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19602440

RESUMO

In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670-1673]. Here, we report the 1.55 A crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with alpha/beta structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Thermus thermophilus/enzimologia , Vitamina K 2/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
13.
J Struct Biol ; 166(1): 32-7, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19135534

RESUMO

Interactions of Bcl-2 family proteins play a regulatory role in mitochondrial apoptosis. The pro-apoptotic protein Bak resides in the outer mitochondrial membrane, and the formation of Bak homo- or heterodimers is involved in the regulation of apoptosis. The previously reported structure of the human Bak protein (residues Glu16-Gly186) revealed that a zinc ion was coordinated with two pairs of Asp160 and His164 residues from the symmetry-related molecules. This zinc-dependent homodimer was regarded as an anti-apoptotic dimer. In the present study, we determined the crystal structure of the human Bak residues Ser23-Asn185 at 2.5A, and found a distinct type of homodimerization through Cys166 disulfide bridging between the symmetry-related molecules. In the two modes of homodimerization, the molecular interfaces are completely different. In the membrane-targeted model of the S-S bridged dimer, the BH3 motifs are too close to the membrane to interact directly with the anti-apoptotic relatives, such as Bcl-x(L). Therefore, the Bak dimer structure reported here may represent a pro-apoptotic mode under oxidized conditions.


Assuntos
Modelos Moleculares , Multimerização Proteica , Proteína Killer-Antagonista Homóloga a bcl-2/química , Sequência de Aminoácidos , Cristalização , Cristalografia por Raios X , Cisteína/química , Cistina/química , Interações Hidrofóbicas e Hidrofílicas , Luz , Dados de Sequência Molecular , Peso Molecular , Oxirredução , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/química , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Recombinantes/química , Espalhamento de Radiação , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Proteína Killer-Antagonista Homóloga a bcl-2/genética
14.
J Biol Chem ; 283(28): 19657-64, 2008 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-18477562

RESUMO

Cyclic nucleotide phosphodiesterases (PDEs) catalyze the degradation of the cyclic nucleotides cAMP and cGMP, which are important second messengers. Five of the 11 mammalian PDE families have tandem GAF domains at their N termini. PDE10A may be the only mammalian PDE for which cAMP is the GAF domain ligand, and it may be allosterically stimulated by cAMP. PDE10A is highly expressed in striatal medium spiny neurons. Here we report the crystal structure of the C-terminal GAF domain (GAF-B) of human PDE10A complexed with cAMP at 2.1-angstroms resolution. The conformation of the PDE10A GAF-B domain monomer closely resembles those of the GAF domains of PDE2A and the cyanobacterium Anabaena cyaB2 adenylyl cyclase, except for the helical bundle consisting of alpha1, alpha2, and alpha5. The PDE10A GAF-B domain forms a dimer in the crystal and in solution. The dimerization is mainly mediated by hydrophobic interactions between the helical bundles in a parallel arrangement, with a large buried surface area. In the PDE10A GAF-B domain, cAMP tightly binds to a cNMP-binding pocket. The residues in the alpha3 and alpha4 helices, the beta6 strand, the loop between 3(10) and alpha4, and the loop between alpha4 and beta5 are involved in the recognition of the phosphate and ribose moieties. This recognition mode is similar to those of the GAF domains of PDE2A and cyaB2. In contrast, the adenine base is specifically recognized by the PDE10A GAF-B domain in a unique manner, through residues in the beta1 and beta2 strands.


Assuntos
Diester Fosfórico Hidrolases/química , Anabaena/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/fisiologia , Cristalografia por Raios X , AMP Cíclico/química , AMP Cíclico/genética , AMP Cíclico/metabolismo , GMP Cíclico/química , GMP Cíclico/genética , GMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/química , Nucleotídeo Cíclico Fosfodiesterase do Tipo 2/metabolismo , Dimerização , Humanos , Neurônios/enzimologia , Diester Fosfórico Hidrolases/metabolismo , Estrutura Secundária de Proteína/fisiologia , Estrutura Terciária de Proteína/fisiologia , Homologia Estrutural de Proteína , Córtex Visual/enzimologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-18084077

RESUMO

The gene encoding TTHA1544 is a singleton found in the Thermus thermophilus HB8 genome and encodes a 131-amino-acid protein. The crystal structure of TTHA1544 has been determined at 2.0 A resolution by the single-wavelength anomalous dispersion method in order to elucidate its function. There are two molecules in the asymmetric unit. Each molecule consists of four alpha-helices and six beta-strands, with the beta-strands composing a central beta-sheet. A structural homology search revealed that the overall structure of TTHA1544 resembles the alpha/beta-hydrolase fold, although TTHA1544 lacks the catalytic residues of a hydrolase. These results suggest that TTHA1544 represents the minimized alpha/beta-hydrolase fold and that an additional component would be required for its activity.


Assuntos
Hidrolases/química , Hidrolases/metabolismo , Dobramento de Proteína , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Humanos , Hidrolases/genética , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia Estrutural de Proteína , Thermus thermophilus/genética
16.
Acta Crystallogr D Biol Crystallogr ; 63(Pt 3): 357-65, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17327673

RESUMO

Glyoxylate reductase catalyzes the NAD(P)H-linked reduction of glyoxylate to glycolate. Here, the 1.7 A crystal structure of glyoxylate reductase from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 complexed with nicotinamide adenine dinucleotide phosphate [NADP(H)] determined by the single-wavelength anomalous dispersion (SAD) method is reported. The monomeric structure comprises the two domains typical of NAD(P)-dependent dehydrogenases: the substrate-binding domain (SBD) and the nucleotide-binding domain (NBD). The crystal structure and analytical ultracentrifugation results revealed dimer formation. In the NADP(H)-binding site, the pyrophosphate moiety and the 2'-phosphoadenosine moiety are recognized by the glycine-rich loop (residues 157-162) and by loop residues 180-182, respectively. Furthermore, the present study revealed that P. horikoshii glyoxylate reductase contains aromatic clusters and has a larger number of ion pairs and a lower percentage of hydrophobic accessible surface area than its mesophilic homologues, suggesting its thermostability mechanism.


Assuntos
Oxirredutases do Álcool/química , Proteínas Arqueais/química , NADP/química , Pyrococcus horikoshii/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Dimerização , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência
17.
J Biol Chem ; 281(42): 31843-53, 2006 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-16928684

RESUMO

Rap2-interacting protein x (RPIPx) is a homolog of RPIP8, a specific effector of Rap2 GTPase. The N-terminal region of RPIP8, which contains the RUN domain, interacts with Rap2. Using cell-free synthesis and NMR, we determined that the region encompassing residues 83-255 of mouse RPIPx, which is 40-residues larger than the predicted RUN domain (residues 113-245), is the minimum fragment that forms a correctly folded protein. This fragment, the RPIPx RUN domain, interacted specifically with Rap2B in vitro in a nucleotide-dependent manner. The crystal structure of the RPIPx RUN domain was determined at 2.0 A of resolution by the multiwavelength anomalous dispersion (MAD) method. The RPIPx RUN domain comprises eight anti-parallel alpha-helices, which form an extensive hydrophobic core, followed by an extended segment. The residues in the core region are highly conserved, suggesting the conservation of the RUN domain-fold among the RUN domain-containing proteins. The residues forming a positively charged surface are conserved between RPIP8 and its homologs, suggesting that this surface is important for Rap2 binding. In the crystal the putative Rap2 binding site of the RPIPx RUN domain interacts with the extended segment in a segment-swapping manner.


Assuntos
Proteínas de Transporte/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , DNA Complementar/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas rap de Ligação ao GTP/química
18.
Protein Sci ; 14(7): 1888-93, 2005 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-15937287

RESUMO

The enhancer of rudimentary gene, e(r), of Drosophila melanogaster encodes an enhancer of rudimentary (ER) protein with functions implicated in pyrimidine biosynthesis and the cell cycle. The ER homolog (ERH) is highly conserved among vertebrates, invertebrates, and plants. Xenopus laevis ERH was reported to be a transcriptional repressor. Here we report the 2.1 Angstroms crystal structure of murine ERH (Protein Data Bank ID 1WZ7), determined by the multiwavelength anomalous dispersion (MAD) method. The monomeric structure of ERH comprises a single domain consisting of three alpha-helices and four beta-strands, which is a novel fold. In the crystal structure, ERH assumes a dimeric structure, through interactions between the beta-sheet regions. The formation of an ERH dimer is consistent with the results of analytical ultracentrifugation. The residues at the core region and at the dimer interface are highly conserved, suggesting the conservation of the dimer formation as well as the monomer fold. The long flexible loop (44 approximately 53) is also significantly conserved, suggesting that this loop region may be important for the functions of ERH. In addition, the putative phosphorylation sites are located at the start of the beta2-strand (Thr18) and at the start of the alpha1-helix (Ser24), implying that the phosphorylation might cause some structural changes.


Assuntos
Proteínas de Ciclo Celular/química , Modelos Moleculares , Conformação Proteica , Fatores de Transcrição/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sequência Conservada , Cristalografia por Raios X , Dimerização , Camundongos , Dados de Sequência Molecular , Fosforilação , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
19.
Artigo em Inglês | MEDLINE | ID: mdl-16508081

RESUMO

The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 A, beta = 106.8 degrees. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 A and refined to an R factor of 16.8% (Rfree = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight beta-strands and seven alpha-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNAPro deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%).


Assuntos
Aeropyrum/enzimologia , Aminoacil-tRNA Sintetases/química , Sequência de Aminoácidos , Aminoacil-tRNA Sintetases/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sequência Conservada , Cristalografia por Raios X , Haemophilus influenzae/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA