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1.
J Biol Chem ; 300(3): 105669, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38272221

RESUMO

The mitotic spindle contains many bundles of microtubules (MTs) including midzones and kinetochore fibers, but little is known about how bundled structures are formed. Here, we show that the chromosomal passenger complex (CPC) purified from Escherichia coli undergoes liquid-liquid demixing in vitro. An emergent property of the resultant condensates is to generate parallel MT bundles when incubated with free tubulin and GTP in vitro. We demonstrate that MT bundles emerge from CPC droplets with protruding minus ends that then grow into long and tapered MT structures. During this growth, we found that the CPC in these condensates apparently reorganize to coat and bundle the resulting MT structures. CPC mutants attenuated for liquid-liquid demixing or MT binding prevented the generation of parallel MT bundles in vitro and reduced the number of MTs present at spindle midzones in HeLa cells. Our data demonstrate that an in vitro biochemical activity to produce MT bundles emerges after the concentration of the CPC and provides models for how cells generate parallel-bundled MT structures that are important for the assembly of the mitotic spindle. Moreover, these data suggest that cells contain MT-organizing centers that generate MT bundles that emerge with the opposite polarity from centrosomes.


Assuntos
Cromossomos , Microtúbulos , Fuso Acromático , Humanos , Células HeLa , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose , Fuso Acromático/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Animais , Xenopus laevis
2.
Biochemistry ; 62(3): 808-823, 2023 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-36625854

RESUMO

3-Ketosteroid Δ1-dehydrogenases (KstD) are important microbial flavin enzymes that initiate the metabolism of steroid ring A and find application in the synthesis of steroid drugs. We present a structure of the KstD from Sterolibacterium denitrificans (AcmB), which contains a previously uncharacterized putative membrane-associated domain and extended proton-relay system. The experimental and theoretical studies show that the steroid Δ1-dehydrogenation proceeds according to the Ping-Pong bi-bi kinetics and a two-step base-assisted elimination (E2cB) mechanism. The mechanism is validated by evaluating the experimental and theoretical kinetic isotope effect for deuterium-substituted substrates. The role of the active-site residues is quantitatively assessed by point mutations, experimental activity assays, and QM/MM MD modeling of the reductive half-reaction (RHR). The pre-steady-state kinetics also reveals that the low pH (6.5) optimum of AcmB is dictated by the oxidative half-reaction (OHR), while the RHR exhibits a slight optimum at the pH usual for the KstD family of 8.5. The modeling confirms the origin of the enantioselectivity of C2-H activation and substrate specificity for Δ4-3-ketosteroids. Finally, the cholest-4-en-3-one turns out to be the best substrate of AcmB in terms of ΔG of binding and predicted rate of dehydrogenation.


Assuntos
Oxirredutases , Prótons , Oxirredutases/metabolismo , Catálise , Esteroides/metabolismo , Mutagênese , Cetosteroides , Cinética , Especificidade por Substrato
3.
J Struct Biol ; 202(3): 229-235, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29408320

RESUMO

Thebaine 6-O-demethylase (T6ODM) from Papaver somniferum (opium poppy), which belongs to the non-heme 2-oxoglutarate/Fe(II)-dependent dioxygenases (ODD) family, is a key enzyme in the morphine biosynthesis pathway. Initially, T6ODM was characterized as an enzyme catalyzing O-demethylation of thebaine to neopinone and oripavine to morphinone. However, the substrate range of T6ODM was recently expanded to a number of various benzylisoquinoline alkaloids. Here, we present crystal structures of T6ODM in complexes with 2-oxoglutarate (T6ODM:2OG, PDB: 5O9W) and succinate (T6ODM:SIN, PDB: 5O7Y). Both metal and 2OG binding sites display similarity to other proteins from the ODD family, but T6ODM is characterized by an exceptionally large substrate binding cavity, whose volume can partially explain the promiscuity of this enzyme. Moreover, the size of the cavity allows for binding of multiple molecules at once, posing a question about the substrate-driven specificity of the enzyme.


Assuntos
Oxirredutases O-Desmetilantes/ultraestrutura , Papaver/enzimologia , Tebaína/química , Cristalografia por Raios X , Ácidos Cetoglutáricos/química , Metilação , Morfina/biossíntese , Morfina/química , Oxirredutases O-Desmetilantes/química , Papaver/química , Ácido Succínico/química
4.
Protein Expr Purif ; 134: 47-62, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28343996

RESUMO

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.


Assuntos
Proteínas de Bactérias , Coenzimas , Escherichia coli/metabolismo , Expressão Gênica , Metaloproteínas , Chaperonas Moleculares , Nitrosomonadaceae/genética , Pteridinas , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Coenzimas/biossíntese , Coenzimas/química , Coenzimas/genética , Coenzimas/isolamento & purificação , Escherichia coli/química , Escherichia coli/genética , Metaloproteínas/biossíntese , Metaloproteínas/química , Metaloproteínas/genética , Metaloproteínas/isolamento & purificação , Chaperonas Moleculares/biossíntese , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Cofatores de Molibdênio , Nitrosomonadaceae/metabolismo , Pteridinas/química , Pteridinas/isolamento & purificação , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
5.
J Struct Funct Genomics ; 17(1): 1-16, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26935210

RESUMO

The period 2000-2015 brought the advent of high-throughput approaches to protein structure determination. With the overall funding on the order of $2 billion (in 2010 dollars), the structural genomics (SG) consortia established worldwide have developed pipelines for target selection, protein production, sample preparation, crystallization, and structure determination by X-ray crystallography and NMR. These efforts resulted in the determination of over 13,500 protein structures, mostly from unique protein families, and increased the structural coverage of the expanding protein universe. SG programs contributed over 4400 publications to the scientific literature. The NIH-funded Protein Structure Initiatives alone have produced over 2000 scientific publications, which to date have attracted more than 93,000 citations. Software and database developments that were necessary to handle high-throughput structure determination workflows have led to structures of better quality and improved integrity of the associated data. Organized and accessible data have a positive impact on the reproducibility of scientific experiments. Most of the experimental data generated by the SG centers are freely available to the community and has been utilized by scientists in various fields of research. SG projects have created, improved, streamlined, and validated many protocols for protein production and crystallization, data collection, and functional analysis, significantly benefiting biological and biomedical research.


Assuntos
Biologia Computacional/métodos , Bases de Dados de Proteínas , Genômica/métodos , Conformação Proteica , Proteínas/química , Proteômica/métodos , Pesquisa Biomédica/métodos , Pesquisa Biomédica/estatística & dados numéricos , Pesquisa Biomédica/tendências , Biologia Computacional/estatística & dados numéricos , Biologia Computacional/tendências , Cristalografia por Raios X , Humanos , Espectroscopia de Ressonância Magnética , Proteínas/genética , Proteínas/metabolismo
6.
Elife ; 132024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38456462

RESUMO

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.


Assuntos
Proteínas de Ciclo Celular , Separação de Fases , Proteínas de Ciclo Celular/genética , Cromossomos , Mitose , Citoesqueleto , Segregação de Cromossomos , Aurora Quinase B/genética
7.
Structure ; 32(6): 725-738.e8, 2024 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-38518780

RESUMO

Entry of Salmonella into host enterocytes relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a 1:2 stoichiometry with sub-nanomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the groove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved by a combination of fast association via the core and very slow dissociation dictated by the arm. Similar to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by actin depolymerizing factor (ADF)/cofilin, which correlated with increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.


Assuntos
Actinas , Proteínas de Bactérias , Faloidina , Fosfatos , Ligação Proteica , Actinas/metabolismo , Actinas/química , Faloidina/metabolismo , Faloidina/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Fosfatos/metabolismo , Fosfatos/química , Microscopia Crioeletrônica , Modelos Moleculares , Sítios de Ligação , Humanos , Fatores de Despolimerização de Actina/metabolismo , Fatores de Despolimerização de Actina/química , Salmonella typhimurium/metabolismo , Proteínas dos Microfilamentos
8.
bioRxiv ; 2023 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-37292983

RESUMO

The physical basis of phase separation is thought to consist of the same types of bonds that specify conventional macromolecular interactions yet is unsatisfyingly often referred to as 'fuzzy'. Gaining clarity on the biogenesis of membraneless cellular compartments is one of the most demanding challenges in biology. Here, we focus on the chromosome passenger complex (CPC), that forms a chromatin body that regulates chromosome segregation in mitosis. Within the three regulatory subunits of the CPC implicated in phase separation - a heterotrimer of INCENP, Survivin, and Borealin - we identify the contact regions formed upon droplet formation using hydrogen/deuterium-exchange mass spectrometry (HXMS). These contact regions correspond to some of the interfaces seen between individual heterotrimers within the crystal lattice they form. A major contribution comes from specific electrostatic interactions that can be broken and reversed through initial and compensatory mutagenesis, respectively. Our findings reveal structural insight for interactions driving liquid-liquid demixing of the CPC. Moreover, we establish HXMS as an approach to define the structural basis for phase separation.

9.
bioRxiv ; 2023 Dec 26.
Artigo em Inglês | MEDLINE | ID: mdl-38234808

RESUMO

Entry of Salmonella into host enterocytes strictly relies on its pathogenicity island 1 effector SipA. We found that SipA binds to F-actin in a unique mode in a 1:2 stoichiometry with picomolar affinity. A cryo-EM reconstruction revealed that SipA's globular core binds at the grove between actin strands, whereas the extended C-terminal arm penetrates deeply into the inter-strand space, stabilizing F-actin from within. The unusually strong binding of SipA is achieved via a combination of fast association via the core and very slow dissociation dictated by the arm. Similarly to Pi, BeF3, and phalloidin, SipA potently inhibited actin depolymerization by ADF/cofilin, which correlated with the increased filament stiffness, supporting the hypothesis that F-actin's mechanical properties contribute to the recognition of its nucleotide state by protein partners. The remarkably strong binding to F-actin maximizes the toxin's effects at the injection site while minimizing global influence on the cytoskeleton and preventing pathogen detection by the host cell.

10.
Mol Biol Cell ; 33(9): br15, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35653296

RESUMO

The Chromosome Passenger Complex (CPC) generates chromosome autonomous signals that regulate mitotic events critical for genome stability. Tip60 is a lysine acetyltransferase that is a tumor suppressor and is targeted for proteasomal degradation by oncogenic papilloma viruses. Mitotic regulation requires the localization of the CPC to inner centromeres, which is driven by the Haspin kinase phosphorylating histone H3 on threonine 3 (H3T3ph). Here we describe how Tip60 acetylates histone H3 at lysine 4 (H3K4ac) to block both the H3T3ph writer and the reader to ensure that this mitotic signaling cannot begin before prophase. Specifically, H3K4ac inhibits Haspin phosphorylation of H3T3 and prevents binding of the Survivin subunit to H3T3ph. Tip60 acetylates H3K4 during S/G2 at centromeres. Inhibition of Tip60 allows the CPC to bind centromeres in G2 cells, and targeting of Tip60 to centromeres prevents CPC localization in mitosis. The H3K4ac mark is removed in prophase by HDAC3 to initiate the CPC localization cascade. Together, our results suggest that Tip60 and HDAC3 temporally control H3K4 acetylation to precisely time the targeting of the CPC to inner centromeres.


Assuntos
Histonas , Proteínas Serina-Treonina Quinases , Acetilação , Centrômero/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose , Fosforilação , Treonina/genética , Treonina/metabolismo
11.
Cell Mol Biol Lett ; 16(4): 625-37, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21910090

RESUMO

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the "leading front" formation during cancer invasion.


Assuntos
Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Próstata/metabolismo , Neoplasias da Próstata , Migração Transendotelial e Transepitelial , Comunicação Celular , Linhagem Celular Tumoral , Movimento Celular , Conexina 43/antagonistas & inibidores , Conexina 43/genética , Junções Comunicantes/genética , Junções Comunicantes/patologia , Expressão Gênica/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Ácido Glicirretínico/farmacologia , Humanos , Masculino , Membranas Artificiais , Invasividade Neoplásica , Porosidade , Próstata/efeitos dos fármacos , Próstata/patologia , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA Interferente Pequeno/farmacologia
12.
FEBS J ; 288(4): 1366-1386, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32592631

RESUMO

Kanamycin A is an aminoglycoside antibiotic isolated from Streptomyces kanamyceticus and used against a wide spectrum of bacteria, including Mycobacterium tuberculosis. Biosynthesis of kanamycin involves an oxidative deamination step catalyzed by kanamycin B dioxygenase (KanJ), thereby the C2' position of kanamycin B is transformed into a keto group upon release of ammonia. Here, we present for the first time, structural models of KanJ with several ligands, which along with the results of ITC binding assays and HPLC activity tests explain substrate specificity of the enzyme. The large size of the binding pocket suggests that KanJ can accept a broad range of substrates, which was confirmed by activity tests. Specificity of the enzyme with respect to its substrate is determined by the hydrogen bond interactions between the methylamino group of the antibiotic and highly conserved Asp134 and Cys150 as well as between hydroxyl groups of the substrate and Asn120 and Gln80. Upon antibiotic binding, the C terminus loop is significantly rearranged and Gln80 and Asn120, which are directly involved in substrate recognition, change their conformations. Based on reaction energy profiles obtained by density functional theory (DFT) simulations, we propose a mechanism of ketone formation involving the reactive FeIV  = O and proceeding either via OH rebound, which yields a hemiaminal intermediate or by abstraction of two hydrogen atoms, which leads to an imine species. At acidic pH, the latter involves a lower barrier than the OH rebound, whereas at basic pH, the barrier leading to an imine vanishes completely. DATABASES: Structural data are available in PDB database under the accession numbers: 6S0R, 6S0T, 6S0U, 6S0W, 6S0V, 6S0S. Diffraction images are available at the Integrated Resource for Reproducibility in Macromolecular Crystallography at http://proteindiffraction.org under DOIs: 10.18430/m36s0t, 10.18430/m36s0u, 10.18430/m36s0r, 10.18430/m36s0s, 10.18430/m36s0v, 10.18430/m36s0w. A data set collection of computational results is available in the Mendeley Data database under DOI: 10.17632/sbyzssjmp3.1 and in the ioChem-BD database under DOI: 10.19061/iochem-bd-4-18.


Assuntos
Proteínas de Bactérias/metabolismo , Dioxigenases/metabolismo , Canamicina/análogos & derivados , Streptomyces/enzimologia , Aminoglicosídeos/química , Aminoglicosídeos/metabolismo , Antibacterianos/química , Antibacterianos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biocatálise , Sequência de Carboidratos , Domínio Catalítico , Cristalografia por Raios X , Dioxigenases/química , Dioxigenases/genética , Canamicina/química , Canamicina/metabolismo , Cinética , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Ligação Proteica , Conformação Proteica , Streptomyces/genética , Especificidade por Substrato
13.
Methods Mol Biol ; 2199: 209-236, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33125653

RESUMO

Efficient and comprehensive data management is an indispensable component of modern scientific research and requires effective tools for all but the most trivial experiments. The LabDB system developed and used in our laboratory was originally designed to track the progress of a structure determination pipeline in several large National Institutes of Health (NIH) projects. While initially designed for structural biology experiments, its modular nature makes it easily applied in laboratories of various sizes in many experimental fields. Over many years, LabDB has transformed into a sophisticated system integrating a range of biochemical, biophysical, and crystallographic experimental data, which harvests data both directly from laboratory instruments and through human input via a web interface. The core module of the system handles many types of universal laboratory management data, such as laboratory personnel, chemical inventories, storage locations, and custom stock solutions. LabDB also tracks various biochemical experiments, including spectrophotometric and fluorescent assays, thermal shift assays, isothermal titration calorimetry experiments, and more. LabDB has been used to manage data for experiments that resulted in over 1200 deposits to the Protein Data Bank (PDB); the system is currently used by the Center for Structural Genomics of Infectious Diseases (CSGID) and several large laboratories. This chapter also provides examples of data mining analyses and warnings about incomplete and inconsistent experimental data. These features, together with its capabilities for detailed tracking, analysis, and auditing of experimental data, make the described system uniquely suited to inspect potential sources of irreproducibility in life sciences research.


Assuntos
Biologia Computacional , Sistemas de Gerenciamento de Base de Dados , Bases de Dados de Proteínas , Humanos , Reprodutibilidade dos Testes
14.
Dalton Trans ; 49(14): 4454-4469, 2020 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-32182320

RESUMO

Hyoscyamine 6ß-hydroxylase (H6H) is a bifunctional non-heme 2-oxoglutarate/Fe2+-dependent dioxygenase that catalyzes the two final steps in the biosynthesis of scopolamine. Based on high resolution crystal structures of H6H from Datura metel, detailed information on substrate binding was obtained that provided insights into the onset of the enzymatic process. In particular, the role of two prominent residues was revealed - Glu-116 that interacts with the tertiary amine located on the hyoscyamine tropane moiety and Tyr-326 that forms CH-π hydrogen bonds with the hyoscyamine phenyl ring. The structures were used as the basis for QM/MM calculations that provided an explanation for the regioselectivity of the hydroxylation reaction on the hyoscyamine tropane moiety (C6 vs. C7) and quantified contributions of active site residues to respective barrier heights.


Assuntos
Oxigenases de Função Mista/química , Oxigenases de Função Mista/metabolismo , Teoria Quântica , Escopolamina/metabolismo , Biocatálise , Hidroxilação , Modelos Moleculares , Conformação Molecular , Escopolamina/química , Estereoisomerismo
15.
Nat Cell Biol ; 21(9): 1127-1137, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31481798

RESUMO

The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a chromatin body with unique biochemical activities at the inner centromere.


Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos/fisiologia , Citoesqueleto/metabolismo , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mitose
16.
Nat Protoc ; 13(5): 1062-1090, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29674755

RESUMO

Metals have crucial roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often a source of irreproducibility caused by issues such as pH changes, incorporation of unexpected metals, or oxidization/reduction of the metal. This protocol outlines the guidelines and best practices for characterizing metal-binding sites in protein structures and alerts experimenters to potential pitfalls during the preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence (XRF) spectra, and collecting diffraction data sets above and below the corresponding metal absorption edges. This protocol should allow experimenters to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecule samples as described in this protocol should enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within a period of 7-190 d, most of which (2-180 d) is devoted to growing the crystal. The protocol should be readily understandable to structural biologists, particularly protein crystallographers with an intermediate level of experience.


Assuntos
Sítios de Ligação , Cristalografia por Raios X/métodos , Metais/metabolismo , Proteínas/química , Proteínas/metabolismo , Ligação Proteica
17.
J Inorg Biochem ; 173: 28-43, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28482186

RESUMO

Steroid C25 dehydrogenase (S25DH) is a molybdenum-containing oxidoreductase isolated from the anaerobic Sterolibacterium denitrificans Chol-1S. S25DH is classified as 'EBDH-like' enzyme (EBDH, ethylbenzene dehydrogenase) and catalyzes the introduction of an OH group to the C25 atom of a sterol aliphatic side-chain. Due to its regioselectivity, S25DH is proposed as a catalyst in production of pharmaceuticals: calcifediol or 25-hydroxycholesterol. The aim of presented research was to obtain structural model of catalytic subunit α and investigate the reaction mechanism of the O2-independent tertiary carbon atom activation. Based on homology modeling and theoretical calculations, a S25DH α subunit model was for the first time characterized and compared to other S25DH-like isoforms. The molecular dynamics simulations of the enzyme-substrate complexes revealed two stable binding modes of a substrate, which are stabilized predominantly by van der Waals forces in the hydrophobic substrate channel. However, H-bond interactions involving polar residues with C3=O/C3-OH in the steroid ring appear to be responsible for positioning the substrate. These results may explain the experimental kinetic results which showed that 3-ketosterols are hydroxylated 5-10-fold faster than 3-hydroxysterols. The reaction mechanism was studied using QM:MM and QM-only cluster models. The postulated mechanism involves homolytic CH cleavage by the MoO ligand, giving rise to a radical intermediate with product obtained in an OH rebound process. The hypothesis was supported by kinetic isotopic effect (KIE) experiments involving 25,26,26,26-[2H]-cholesterol (4.5) and the theoretically predicted intrinsic KIE (7.0-7.2). Finally, we have demonstrated that the recombinant S25DH-like isoform catalyzes the same reaction as S25DH.


Assuntos
Isoenzimas/metabolismo , Oxirredutases/metabolismo , Anaerobiose , Domínio Catalítico , Bactérias Gram-Negativas/enzimologia , Ligação de Hidrogênio , Hidroxilação , Hidroxiesteroides/metabolismo , Isoenzimas/química , Cetosteroides/metabolismo , Cinética , Oxirredutases/química , Rhodocyclaceae/enzimologia , Especificidade por Substrato
18.
Protein Sci ; 25(3): 720-33, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26660914

RESUMO

The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.


Assuntos
Cristalização/métodos , Proteínas/química , Acetiltransferases/química , Acetiltransferases/isolamento & purificação , Animais , Artefatos , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/isolamento & purificação , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Proteínas/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Reprodutibilidade dos Testes , Fator sigma/química , Fator sigma/isolamento & purificação , Staphylococcus aureus/química , Survivina , Xenopus/metabolismo , Proteínas de Xenopus/química
19.
Mol Biol Cell ; 23(8): 1457-66, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22357620

RESUMO

Survivin, a subunit of the chromosome passenger complex (CPC), binds the N-terminal tail of histone H3, which is phosphorylated on T3 by Haspin kinase, and localizes the complex to the inner centromeres. We used x-ray crystallography to determine the residues of Survivin that are important in binding phosphomodified histone H3. Mutation of amino acids that interact with the histone N-terminus lowered in vitro tail binding affinity and reduced CPC recruitment to the inner centromere in cells, validating our solved structures. Phylogenetic analysis shows that nonmammalian vertebrates have two Survivin paralogues, which we name class A and B. A distinguishing feature of these paralogues is an H-to-R change in an amino acid that interacts with the histone T3 phosphate. The binding to histone tails of the human class A paralogue, which has a histidine at this position, is sensitive to changes around physiological pH, whereas Xenopus Survivin class B is less so. Our data demonstrate that Survivin paralogues have different characteristics of phosphospecific binding to threonine-3 of histone H3, providing new insight into the biology of the inner centromere.


Assuntos
Centrômero/metabolismo , Histonas/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Células HeLa , Histonas/química , Humanos , Proteínas Inibidoras de Apoptose/química , Fosforilação , Estrutura Terciária de Proteína , Alinhamento de Sequência , Survivina
20.
Science ; 330(6001): 231-5, 2010 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-20705812

RESUMO

Aurora B is a component of the chromosomal passenger complex (CPC) required for correct spindle-kinetochore attachments during chromosome segregation and for cytokinesis. The chromatin factors that recruit the CPC to centromeres are unknown, however. Here we show that phosphorylation of histone H3 threonine 3 (H3T3ph) by Haspin is necessary for CPC accumulation at centromeres and that the CPC subunit Survivin binds directly to H3T3ph. A nonbinding Survivin-D70A/D71A mutant does not support centromeric CPC concentration, and both Haspin depletion and Survivin-D70A/D71A mutation diminish centromere localization of the kinesin MCAK and the mitotic checkpoint response to taxol. Survivin-D70A/D71A mutation and microinjection of H3T3ph-specific antibody both compromise centromeric Aurora B functions but do not prevent cytokinesis. Therefore, H3T3ph generated by Haspin positions the CPC at centromeres to regulate selected targets of Aurora B during mitosis.


Assuntos
Centrômero/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Aurora Quinase B , Aurora Quinases , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose , Peptídeos e Proteínas de Sinalização Intracelular/genética , Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Mutação , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Proteínas Recombinantes/metabolismo , Fuso Acromático/metabolismo , Survivina , Suínos , Treonina/metabolismo , Xenopus
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