RESUMO
The autosomal dominant, giant-platelet disorders, May-Hegglin anomaly (MHA; MIM 155100), Fechtner syndrome (FTNS; MIM 153640) and Sebastian syndrome (SBS), share the triad of thrombocytopenia, large platelets and characteristic leukocyte inclusions ('Döhle-like' bodies). MHA and SBS can be differentiated by subtle ultrastructural leukocyte inclusion features, whereas FTNS is distinguished by the additional Alport-like clinical features of sensorineural deafness, cataracts and nephritis. The similarities between these platelet disorders and our recent refinement of the MHA (ref. 6) and FTNS (ref. 7) disease loci to an overlapping region of 480 kb on chromosome 22 suggested that all three disorders are allelic. Among the identified candidate genes is the gene encoding nonmuscle myosin heavy chain 9 (MYH9; refs 8-10), which is expressed in platelets and upregulated during granulocyte differentiation. We identified six MYH9 mutations (one nonsense and five missense) in seven unrelated probands from MHA, SBS and FTNS families. On the basis of molecular modelling, the two mutations affecting the myosin head were predicted to impose electrostatic and conformational changes, whereas the truncating mutation deleted the unique carboxy-terminal tailpiece. The remaining missense mutations, all affecting highly conserved coiled-coil domain positions, imparted destabilizing electrostatic and polar changes. Thus, our results suggest that mutations in MYH9 result in three megakaryocyte/platelet/leukocyte syndromes and are important in the pathogenesis of sensorineural deafness, cataracts and nephritis.
Assuntos
Transtornos Plaquetários/genética , Leucócitos/patologia , Proteínas Motores Moleculares , Mutação , Cadeias Pesadas de Miosina/genética , Alelos , Sequência de Aminoácidos , Animais , Transtornos Plaquetários/patologia , Catarata/genética , Galinhas , Cromossomos Humanos Par 22 , Cristalografia por Raios X , Citoplasma/metabolismo , Genótipo , Perda Auditiva Neurossensorial/genética , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Músculo Liso/metabolismo , Mutação de Sentido Incorreto , Cadeias Pesadas de Miosina/química , Miosinas/química , Miosinas/genética , Nefrite/genética , Neutrófilos/patologia , Neutrófilos/ultraestrutura , Fenótipo , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Síndrome , Trombocitopenia/genéticaRESUMO
BACKGROUND: Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. RESULTS: Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. CONCLUSIONS: Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli , Tiossulfato Sulfurtransferase/química , Sequência de Aminoácidos , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Família Multigênica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Sulfurtransferases/químicaRESUMO
BACKGROUND: The process of guanosine 5'-diphosphate L-fucose (GDP-L-fucose) biosynthesis is conserved throughout evolution from prokaryotes to man. In animals, GDP-L-fucose is the substrate of fucosyltransferases that participate in the biosynthesis and remodeling of glycoconjugates, including ABH blood group and Lewis-system antigens. The 'de novo' pathway of GDP-L-fucose biosynthesis from GDP-D-mannose involves a GDP-D-mannose 4,6 dehydratase (GMD) and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER). Neither of the catalytic mechanisms nor the three-dimensional structures of the two enzymes has been elucidated yet. The severe leukocyte adhesion deficiency (LAD) type II genetic syndrome is known to result from deficiencies in this de novo pathway. RESULTS: The crystal structures of apo- and holo-GMER have been determined at 2.1 A and 2.2 A resolution, respectively. Each subunit of the homodimeric (2 x 34 kDa) enzyme is composed of two domains. The N-terminal domain, a six-stranded Rossmann fold, binds NADP+; the C-terminal domain (about 100 residues) displays an alpha/beta topology. NADP+ interacts with residues Arg12 and Arg36 at the adenylic ribose phosphate; moreover, a protein loop based on the Gly-X-X-Gly-X-X-Gly motif (where X is any amino acid) stabilizes binding of the coenzyme diphosphate bridge. The nicotinamide and the connected ribose ring are located close to residues Ser107, Tyr136 and Lys140, the putative GMER active-site center. CONCLUSIONS: The GMER fold is reminiscent of that observed for UDP-galactose epimerase (UGE) from Escherichia coli. Consideration of the enzyme fold and of its main structural features allows assignment of GMER to the reductase-epimerase-dehydrogenase (RED) enzyme homology superfamily, to which short-chain dehydrogenase/reductases (SDRs) also belong. The location of the NADP+ nicotinamide ring at an interdomain cleft is compatible with substrate binding in the C-terminal domain.
Assuntos
Carboidratos Epimerases/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimologia , Guanosina Difosfato Fucose/biossíntese , Cetona Oxirredutases , Complexos Multienzimáticos , Desidrogenase do Álcool de Açúcar/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Carboidratos Epimerases/química , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , NADP/metabolismo , Conformação Proteica , Dobramento de Proteína , Homologia de Sequência de Aminoácidos , Desidrogenase do Álcool de Açúcar/químicaRESUMO
The amino acid sequences of ten globin chain tertiary structures were aligned and structurally equivalenced by spatial superposition of main-chain C alpha atoms. A search was then performed for structurally equivalent residue pairs that were buried in the protein core and that had mutated but maintained similar unmutated environments. Residues with atoms in contact with such central residue pairs define their environments. Such examples of point mutations would represent in vivo site-directed mutagenesis as would be observed in evolution. A search for mutated but exposed equivalent central residues was also performed. The constraints placed on the characteristics of the mutated residues (e.g., side-chain volume, polarity, radius of gyration) allow suggestions for the evolutionary modes of protein core and surface development as well as residue substitution guidelines to maintain structural stability in protein engineering and design.
Assuntos
Evolução Biológica , Globinas/genética , Mutação , Conformação Proteica , Sequência de Aminoácidos , Animais , Sistemas de Informação , Substâncias Macromoleculares , Matemática , Modelos Moleculares , Modelos Teóricos , Dados de Sequência MolecularRESUMO
Intra-molecular side-chain:main-chain (sch:mch) and side-chain (sch:sch) hydrogen bonds observed in 44 well refined crystallographic protein structures with non-homologous sequences have been identified, classified and analysed to detect recurring structural patterns. Each observed bond was characterized by the position of its acceptor and donor groups relative to the N and C termini of the particular secondary structure in which they occur and according to their appearance within the same of sequentially separated secondary structures. The role of short-range hydrogen bonds in the formation and stabilization of a secondary structure and the importance of long-range hydrogen bonds as a cohesive force for different structural segments were also examined. It was found that the N terminus of alpha-helices is characterized by recurring sch:mch and sch:sch bonds with elements of the preceding coil segment, while at the C terminus a frequent intra-helix sch:mch hydrogen bond was frequently observed. The residues at or near the beta-strand termini often cross-linked, through hydrogen-bonding, non-sequential coil segments. Coil structures were characterized by recurring, internal sch:mch hydrogen bonding involving small polar side-chain groups situated at or near their N termini (coil N-capping). The significance of hydrogen bonds as formers and stabilizers of a protein fold and the association of its secondary structural units was also considered through an examination of bond density and distribution throughout the protein tertiary structure.
Assuntos
Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas/química , Aminoácidos/química , Ligação de Hidrogênio , Estrutura MolecularRESUMO
The conserved topological structure observed in various molecular families such as globins or cytochromes c allows structural equivalencing of residues in every homologous structure and defines in a coherent way a global alignment in each sequence family. A search was performed for equivalent residue pairs in various topological families that were buried in protein cores or exposed at the protein surface and that had mutated but maintained similar unmutated environments. Amino acid residues with atoms in contact with the mutated residue pairs defined the environment. Matrices of preferred amino acid exchanges were then constructed and preferred or avoided amino acid substitutions deduced. Given the conserved atomic neighborhoods, such natural in vivo substitutions are subject to similar constrains as point mutations performed in site-directed mutagenesis experiments. The exchange matrices should provide guidelines for "safe" amino acid substitutions least likely to disturb the protein structure, either locally or in its overall folding pathway, and most likely to allow probing the structural and functional significance of the substituted site.
Assuntos
Mutagênese Sítio-Dirigida , Proteínas/química , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas/análise , Proteínas/genéticaRESUMO
Conserved structural and functional features of Cu,Zn superoxide dismutase enzymes have been studied by comparison of known three-dimensional structures and analysis of the currently available amino acid sequences. For this purpose, the three-dimensional structures of the bovine, spinach and yeast enzymes have been superimposed and the structure-based sequence alignment of 38 different superoxide dismutases has been produced. The evolutionary tree obtained from the alignment indicates that cytosolic and extracellular enzymes followed independent evolutionary paths, and that horizontal gene transfer, if any, occurred at an early stage in eukaryota evolution. Based on the sequence alignment and on the analysis of clusters of spatially neighboring residues, the conservation/variation of functionally relevant intramolecular interactions has been investigated. Seven alternative residue arrangements have been identified in the upper rim of the active site, which form an important determinant of the electrostatic field at the catalytic center. The total nominal charge of this region is constantly -1 through the phyla. The seven residues which coordinate the two metal ions at the active site are conserved, with only one known exception. Among the residues involved in maintenance of the active site structure, Gly59, Gly80, Gly136 and Gly139 are fully conserved; mutations of Gly42 and Pro64 have been observed, concerted with replacements in their structural surroundings. Coordinated mutations affecting residue pairs which maintain the packing geometry of the Greek-key beta-barrel have been identified. Furthermore, the unique disulfide bridge involving Cys55-Cys144 in eukaryota, shows the alternative Cys50A-Cys144 arrangement in prokaryotic enzymes.
Assuntos
Superóxido Dismutase/química , Sequência de Aminoácidos , Animais , Evolução Biológica , Bovinos , Gráficos por Computador , Cobre/química , Dissulfetos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Superóxido Dismutase/genética , Verduras/enzimologia , Leveduras/enzimologia , Zinco/químicaRESUMO
Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a rhodanese. The crystal structure of the A. vinelandii rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.
Assuntos
Azotobacter vinelandii/enzimologia , Evolução Molecular , Monoéster Fosfórico Hidrolases/química , Sulfurtransferases/química , Tiossulfato Sulfurtransferase/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Catálise , Bovinos , Cristalografia por Raios X , Humanos , Ligação de Hidrogênio , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade Estática , Sulfurtransferases/genética , Sulfurtransferases/metabolismo , Água/metabolismoRESUMO
The functional properties and X-ray structures of five mutant forms of Photobacterium leiognathi Cu,Zn superoxide dismutase carrying single mutations at residues located at the dimer association interface have been investigated. When compared to the wild-type enzyme, the three-dimensional structures of the mutants show structural perturbations limited to the proximity of the mutation sites and substantial identity of active site geometry. Nonetheless, the catalytic rates of all mutants, measured at neutral pH and low ionic strength by pulse radiolysis, are higher than that of the wild-type protein. Such enzymatic activity increase is paralleled by enhanced active site accessibility to external chelating agents, which, in the mutated enzyme, remove more readily the active site copper ion. It is concluded that mutations at the prokaryotic Cu,Zn superoxide dismutase subunit interface can transduce dynamical perturbation to the active site region, promoting substrate active site accessibility. Such long-range intramolecular communication effects have not been extensively described before within the Cu,Zn superoxide dismutase homology family.
Assuntos
Cobre/metabolismo , Mutação/genética , Photobacterium/enzimologia , Superóxido Dismutase/química , Superóxido Dismutase/metabolismo , Substituição de Aminoácidos/genética , Sítios de Ligação , Catálise/efeitos dos fármacos , Quelantes/farmacologia , Cristalografia por Raios X , Dimerização , Ácido Edético/farmacologia , Cinética , Modelos Moleculares , Photobacterium/genética , Estrutura Quaternária de Proteína , Subunidades Proteicas , Radiólise de Impulso , Solventes , Análise Espectral , Superóxido Dismutase/genética , Temperatura , Fatores de TempoRESUMO
The bacterial rpoN operon codes for sigma 54, which is the key sigma factor that, under nitrogen starvation conditions, activates the transcription of genes needed to assimilate ammonia and glutamate. The rpoN operon contains several other open reading frames that are cotranscribed with sigma 54. The product of one of these, the 17.9 kDa protein IIANtr, is homologous to IIA proteins of the phosphoenolpyruvate:sugar phosphotransferase (PTS) system. IIANtr influences the transcription of sigma 54-dependent genes through an unknown mechanism and may thereby provide a regulatory link between carbon and nitrogen metabolism. Here we describe the 2.35 A X-ray structure of Escherichia coli IIANtr. It is the first structure of a IIA enzyme from the fructose-mannitol family of the PTS. The enzyme displays a novel fold characterized by a central mixed parallel/anti-parallel beta-sheet surrounded by six alpha-helices. The active site His73 is situated in a shallow depression on the protein surface.
Assuntos
Proteínas de Bactérias , Proteínas de Ligação a DNA/química , Escherichia coli/química , Conformação Proteica , Transativadores , Fatores de Transcrição , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , RNA Polimerases Dirigidas por DNA/química , Proteínas de Escherichia coli , Modelos Moleculares , Dados de Sequência Molecular , Proteínas PII Reguladoras de Nitrogênio , Sistema Fosfotransferase de Açúcar do Fosfoenolpiruvato/química , RNA Polimerase Sigma 54 , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fator sigma/químicaRESUMO
Prokaryotic Cu,Zn superoxide dismutases are characterized by a distinct quaternary structure, as compared to that of the homologous eukaryotic enzymes. Here we report a newly determined crystal structure of the dimeric Cu,Zn superoxide dismutase from Photobacterium leiognathi (crystallized in space group R32, refined at 2.5 A resolution, R-factor 0.19) and analyse it in comparison with that of the monomeric enzyme from Escherichia coli. The dimeric assembly, observed also in a previously studied monoclinic crystal form of P. leiognathi Cu,Zn superoxide dismutase, is based on a ring-shaped subunit contact region, defining a solvated interface cavity. Three clusters of neighbouring residues play a direct role in the stabilization of the quaternary assembly. The present analysis, extended to the amino acid sequences of the other 11 known prokaryotic Cu,Zn superoxide dismutases, shows that at least in five other prokaryotic enzymes the interface residue clusters are under strong evolutionary constraint, suggesting the attainment of a quaternary structure coincident with that of P. leiognathi Cu,Zn superoxide dismutase. Calculation of electrostatic fields for both the enzymes from E. coli and P. leiognathi shows that the monomeric/dimeric association behaviour displayed by prokaryotic Cu, Zn superoxide dismutases is related to the distribution of surface charged residues. Moreover, Brownian dynamics simulations reproduce closely the observed enzyme:substrate association rates, highlighting the role of the active site neighbouring residues in determining the dismutase catalytic properties.
Assuntos
Evolução Molecular , Photobacterium/enzimologia , Superóxido Dismutase/química , Sequência de Aminoácidos , Animais , Cobre , Cristalografia por Raios X , Dimerização , Dados de Sequência Molecular , Células Procarióticas , Conformação Proteica , Eletricidade Estática , Superóxido Dismutase/classificação , Xenopus laevis , ZincoRESUMO
Azotobacter vinelandii RhdA uses thiosulfate as the only sulfur donor in vitro, and this apparent selectivity seems to be a unique property among the characterized sulfurtransferases. To investigate the basis of substrate recognition in RhdA, we replaced Thr-232 with either Ala or Lys. Thr-232 was the target of this study since the corresponding Lys-249 in bovine rhodanese has been identified as necessary for catalytic sulfur transfer, and replacement of Lys-249 with Ala fully inactivates bovine rhodanese. Both T232K and T232A mutants of RhdA showed significant increase in thiosulfate-cyanide sulfurtransferase activity, and no detectable activity in the presence of 3-mercaptopyruvate as the sulfur donor substrate. Fluorescence measurements showed that wild-type and mutant RhdAs were overexpressed in the persulfurated form, thus conferring to this enzyme the potential of a persulfide sulfur donor compound. RhdA contains a unique sequence stretch around the catalytic cysteine, and the data here presented suggest a possible divergent physiological function of A. vinelandii sulfurtransferase.
Assuntos
Azotobacter vinelandii/enzimologia , Tiossulfato Sulfurtransferase/genética , Treonina/genética , Animais , Bovinos , Humanos , Mutagênese , Células Procarióticas , Sulfurtransferases/classificação , Sulfurtransferases/genética , Sulfurtransferases/metabolismo , Tiossulfato Sulfurtransferase/classificação , Tiossulfato Sulfurtransferase/metabolismo , Treonina/metabolismoRESUMO
We report a new missense mutation (Gly12Arg) [corrected] in exon 1 of the Cu/Zn superoxide dismutase (SOD1) gene in a 67-year-old patient with familial ALS (FALS). The clinical course showed an unusually slow progression. The enzymatic activity of the mutated SOD1 was 80% of normal. At the molecular level, the Gly12Arg [corrected] mutation occurs in a region outside the active site and may lead to local distortion strain in the protein structure.
Assuntos
Esclerose Lateral Amiotrófica/genética , Superóxido Dismutase/genética , Esclerose Lateral Amiotrófica/fisiopatologia , Éxons , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Linhagem , Superóxido Dismutase-1 , Fatores de TempoRESUMO
The objective of this study was to investigate MICA (major histocompatibility complex MHC class I chain-related genes) polymorphisms in an Italian series of patients with juvenile Behcet disease (jBD) and to compare these genetic findings with the high prevalence of inflammatory mucosal disease, which occurs in Western populations. Ten families which included at least 1 affected patient were studied. We genotyped 18 patients (13 children and 5 adults) affected with the complete or incomplete form of jBD comparing the results to those found in a population of 20 apparently healthy individuals. The MICA transmembrane polymorphism was analysed by PCR and polyacrylamide gel electrophoresis. HLA typing was assessed by SSP-PCR technique. Statistical analysis was performed using chi2 based methods. In our series the prevalence of gastrointestinal disease was high (41%). Seven of 10 patients were HLA-B51 positive. MICA A6 allele was present in 70% of probands as compared to 25% of an ethnically matched control population. On the other hand, MICA A5.1 was present in 20% of probands as compared to 60% in controls. Out of 5 A6 homozygotes, 2 probands and 2 affected relatives developed a severe gut inflammatory disease. The study of MICA gene polymorphisms disclosed an independent association with genetic risk for jBD. The combination of MICA A6 and HLA-B51 is the strongest genetic marker for this disease. Homozygous A6 patients seem to develop more severe mucosal gut involvement. This finding sheds light on the role of a receptor for MICA, named NKG2D, presented by natural killer cells, and CD8+, alphabetaT cells and gammadeltaT cells, usually localised in gut mucosa.