RESUMO
The location of isomorphously substituted zinc over eight crystallographically different gallium sites has been determined in a single-crystal study of the gallophosphate ZnULM-5, Ga((16-x))Zn(x)(PO(4))(14)(HPO(4))(2)(OH)(2)F(7), [H(3)N{CH(2)}(6)NH(3)](4), 6H(2)O, in an 11 wavelength experiment, using data from Station 9.8, SRS Daresbury. The measurement of datasets around the K edges of both Ga and Zn, as well as two reference datasets away from each absorption edge, was utilized to selectively exploit dispersive differences of each metal atom type in turn, which allowed the major sites of Zn incorporation to be identified as the metal 1 and 3 sites, M1 and M3. The preferential substitution of Zn at these sites probably arises because they are located in double four-ring (D4R) building units which can relax to accommodate the incorporation of hetero atoms. As the crystal is non-centrosymmetric, with space group P2(1)2(1)2, it was also possible to use anomalous differences to corroborate the results obtained from the dispersive differences. These results were obtained firstly from difference Fourier maps, calculated using a phase set from the refined structure from data measured at the Zr K edge. Also, refined dispersive and anomalous occupancies, on an absolute scale, could be obtained using the program MLPHARE, allowing estimates for the Zn incorporation of approximately 22 and 18 at. % at the M1 and M3 sites to be obtained. In addition, f' and f'' values for Ga and Zn at each wavelength could be estimated both from MLPHARE results, and by refinement in JANA2006. The fully quantitative determinations of the dispersive and anomalous coefficients for Ga and Zn at each wavelength, as well as metal atom occupancies over the eight metal atom sites made use of the CCP4's MLPHARE program as well as SHELXL and JANA2006. The results by these methods agree closely, and JANA2006 allowed the ready determination of standard uncertainties on the occupancy parameters, which were for M1 and M3, 20.6 (3) and 17.2 (3) at %, respectively.
RESUMO
The density-modification procedures incorporated in ACORN, available in the CCP4 package, have proved to be very successful in solving and refining high-resolution crystal structures from very poor starting sets. These can be calculated from a correctly positioned initial fragment containing between 1 and 8% of the scattering power of the total structure. Improvements of ACORN, reported here and incorporated in the program ACORN2, have lowered the size of the fragment required and examples are given of structures solved with only 0.25% of the scattering power in the fragment, which may be a single atom. Applications of ACORN2 to structures with space group P1 have shown the remarkable property that when the starting point is a pair of equal atoms, or even a single atom placed at the origin, the refinement process breaks the centric nature of the initial phases and converges to phases corresponding to one of the two possible enantiomorphs. Examples are given of the application of ACORN2 to the solution and/or refinement of a number of known trial structures and to the refinement of structures when phases are available either from MAD or from a molecular-replacement model.
Assuntos
Álcool Desidrogenase/química , Aldose-Cetose Isomerases/química , Modelos Teóricos , Muramidase/química , Álcool Desidrogenase/metabolismo , Aldose-Cetose Isomerases/metabolismo , Cádmio/química , Cádmio/metabolismo , Cristalização/métodos , Cristalografia por Raios X , Humanos , Muramidase/metabolismo , Conformação Proteica , SoftwareRESUMO
The CD4 antigen is a membrane glycoprotein of T lymphocytes that interacts with major histocompatibility complex class II antigens and is also a receptor for the human immunodeficiency virus. the extracellular portion of CD4 is predicted to fold into four immunoglobulin-like domains. The crystal structure of the third and fourth domains of rat CD4 was solved at 2.8 angstrom resolution and shows that both domains have immunoglobulin folds. Domain 3, however, lacks the disulfide between the beta sheets; this results in an expansion of the domain. There is a difference of 30 degrees in the orientation between domains 3 and 4 when compared with domains 1 and 2. The two CD4 fragment structures provide a basis from which models of the overall receptor can be proposed. These models suggest an extended structure comprising two rigid portions joined by a short and possibly flexible linker region.
Assuntos
Antígenos CD4/química , Sequência de Aminoácidos , Animais , Cristalização , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Ratos , Alinhamento de Sequência , Difração de Raios XRESUMO
Specific protein-ligand interactions are critical for cellular function, and most proteins select their partners with sharp discrimination. However, the oligopeptide-binding protein of Salmonella typhimurium (OppA) binds peptides of two to five amino acid residues without regard to sequence. The crystal structure of OppA reveals a three-domain organization, unlike other periplasmic binding proteins. In OppA-peptide complexes, the ligands are completely enclosed in the protein interior, a mode of binding that normally imposes tight specificity. The protein fulfills the hydrogen bonding and electrostatic potential of the ligand main chain and accommodates the peptide side chains in voluminous hydrated cavities.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Lipoproteínas/metabolismo , Oligopeptídeos/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas de Transporte/química , Cristalografia por Raios X , Ligação de Hidrogênio , Ligantes , Lipoproteínas/química , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Oligopeptídeos/química , Conformação Proteica , Estrutura Secundária de ProteínaRESUMO
Molecular replacement is a possible route to obtaining initial phasing for an unknown structure from a known, structurally related molecule. Recent years have seen an explosive growth in the number of protein structures solved using this technique. Automated packages can make the application quite straightforward. Progress has been made in the placing of fragments of complexes, and in the use of imprecise models from NMR or homology modelling. Such models have necessitated the development of new approaches to rebuilding and refinement.
Assuntos
Modelos Moleculares , Proteínas/química , Animais , Humanos , Espectroscopia de Ressonância MagnéticaRESUMO
During 1994 and 1995, the structures of the serum amyloid P component, the bacterial chaperonin GroEL, the 20S proteasome, the bacterial light-harvesting complexes and the tryptophan operon RNA-binding attenuation protein have been determined. These structures all form circular assemblies in which the individual subunits are related by rotational symmetry. In most cases the circular organization generates a new biophysical property and a specific biological function which have presumably been selected by evolution.
Assuntos
Proteínas de Bactérias , Conformação Proteica , Proteínas/química , Chaperonina 60/química , Cisteína Endopeptidases/química , Humanos , Substâncias Macromoleculares , Complexos Multienzimáticos/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Endopeptidases do Proteassoma , Dobramento de Proteína , Proteínas de Ligação a RNA/química , Componente Amiloide P Sérico/química , Fatores de Transcrição/químicaRESUMO
BACKGROUND: CysB is a tetrameric protein of identical subunits (M(r) = 36,000) which controls the expression of genes associated with the biosynthesis of cysteine in bacteria. CysB is both an activator and a repressor of transcription whose activity is responsive to the inducer N-acetylserine; thiosulphate and sulphide act as anti-inducers. CysB is a member of the LysR family of prokaryotic transcriptional regulatory proteins which share sequence similarities over approximately 280 residues including a putative helix-turn-helix DNA-binding motif at their N terminus. The aims of the present study were to explore further the complex molecular biology and curious ligand binding properties of CysB and to provide structural insights into the LysR family of proteins. RESULTS: The crystal structure of a dimeric chymotryptic fragment of Klebsiella aerogenes CysB comprising residues 88-324, has been solved by multiple isomorphous replacement and multi-crystal averaging and refined against data extending to 1.8 A resolution. The protein comprises two alpha/beta domains (I and II) connected by two short segments of polypeptide. The two domains enclose a cavity lined by polar sidechains, including those of two residues whose mutation is associated with constitutive expression of the cysteine regulon. A sulphate anion and a number of well ordered water molecules have been modelled into discrete electron-density peaks within this cavity. In the dimer, strands beta B from domain I and strands beta G from domain II come together so that a pair of antiparallel symmetry-related 11-stranded twisted beta-pleated sheets is formed. CONCLUSIONS: The overall structure of CysB(88-324) is strikingly similar to those of the periplasmic substrate-binding proteins. A similar fold has also been observed in the cofactor-binding domain of Lac repressor, implying a structural relationship between the Lac repressor and LysR families of proteins. In contrast to Lac repressor, in CysB the twofold axis of symmetry that relates the monomers in the dimer is perpendicular rather than parallel to the long axis of the cofactor-binding domain. This seems likely to place the DNA-binding domains at opposite extremes of the molecule possibly accounting for CysB's extended DNA footprints.
Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Klebsiella pneumoniae/química , Modelos Moleculares , Dados de Sequência Molecular , Peso Molecular , Fragmentos de Peptídeos/química , Conformação Proteica , Alinhamento de SequênciaRESUMO
BACKGROUND: The periplasmic oligopeptide-binding protein OppA has a remarkably broad substrate specificity, binding peptides of two or five amino-acid residues with high affinity, but little regard to sequence. It is therefore an ideal system for studying how different chemical groups can be accommodated in a protein interior. The ability of the protein to bind peptides of different lengths has been studied by co-crystallising it with different ligands. RESULTS: Crystals of OppA from Salmonella typhimurium complexed with the peptides Lys-Lys-Lys (KKK) and Lys-Lys-Lys-Ala (KKKA) have been grown in the presence of uranyl ions which form important crystal contacts. These structures have been refined to 1.4 A and 2.1 A, respectively. The ligands are completely enclosed, their side chains pointing into large hydrated cavities and making few strong interactions with the protein. CONCLUSIONS: Tight peptide binding by OppA arises from strong hydrogen bonding and electrostatic interactions between the protein and the main chain of the ligand. Different basic side chains on the protein form salt bridges with the C terminus of peptide ligands of different lengths.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Lipoproteínas/química , Modelos Moleculares , Oligopeptídeos/metabolismo , Estrutura Terciária de Proteína , Salmonella typhimurium/química , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Antígenos HLA/química , Antígenos HLA/metabolismo , Ligação de Hidrogênio , Ligantes , Lipoproteínas/metabolismo , Dados de Sequência Molecular , Polilisina/metabolismo , Ligação Proteica , Urânio/químicaRESUMO
BACKGROUND: beta-glucosidases occur in a variety of organisms and catalyze the hydrolysis of aryl and alkyl-beta-D-glucosides as well as glucosides with only a carbohydrate moiety (such as cellobiose). The cyanogenic beta-glucosidase from white clover (subsequently referred to as CBG) is responsible for the cleavage of cyanoglucosides. Both CBG and the cyanoglucosides occur within the plant cell wall where they are found in separate compartments and only come into contact when the leaf tissue experiences mechanical damage. This results in the eventual production of hydrogen cyanide which acts as a deterrent to grazing animals. beta-glucosidases have been assigned to particular glycosyl hydrolase families on the basis of sequence similarity; this classification has placed CBG in family 1 (there are a total of over 40 families) for which a three-dimensional structure has so far not been determined. This is the first report of the three-dimensional structure of a glycosyl hydrolase from family 1. RESULTS: The crystal structure of CBG has been determined using multiple isomorphous replacement. The final model has been refined at 2.15 A resolution to an R factor of 18.9%. The overall fold of the molecule is a (beta/alpha)8 [or (alpha/beta)8] barrel (in common with a number of glycosyl hydrolases) with all residues located in a single domain. CONCLUSIONS: Sequence comparisons between beta-glucosidases of the same family show that residues Glu183 and Glu397 are highly conserved. Both residues are positioned at the end of a pocket located at the C terminus of the barrel and have been assigned the respective roles of proton donor and nucleophile on the basis of inhibitor-binding and mutagenesis experiments. These roles are consistent with the environments of the two residues. The pocket itself is typical of a sugar-binding site as it contains a number of charged, aromatic and polar groups. In support of this role, we present crystallographic data on a possible product complex between CBG and glucose, resulting from co-crystallization of the native enzyme with its natural substrate, linamarin.
Assuntos
Cristalografia por Raios X , Fabaceae/enzimologia , Plantas Medicinais , beta-Glucosidase/química , Sítios de Ligação , Glicosídeo Hidrolases/classificação , Modelos Moleculares , Nitrilas/química , Nitrilas/metabolismo , Conformação Proteica , Dobramento de Proteína , beta-Glucosidase/classificação , beta-Glucosidase/metabolismoRESUMO
The explosive increase in the number of published three-dimensionsal structures of macromolecules determined by X-ray analysis places a responsibility on experimentalists, referees and curators of databases to ensure correspondence between the structure parameters and data. Validation tools will evolve as more appropriate statistical techniques and new information, such as that from proteins analysed at atomic resolution, becomes available.
Assuntos
Cristalografia/métodos , Reprodutibilidade dos Testes , Cristalografia por Raios X , Bases de Dados Factuais , Modelos Moleculares , Ressonância Magnética Nuclear BiomolecularRESUMO
BACKGROUND: Infections caused by Candida albicans, a common fungal pathogen of humans, are increasing in incidence, necessitating development of new therapeutic drugs. Secreted aspartic proteinase (SAP) activity is considered an important virulence factor in these infections and might offer a suitable target for drug design. Amongst the various SAP isozymes, the SAP2 gene product is the major form expressed in a number of C. albicans strains. RESULTS: The three-dimensional structures of SAP2 complexed with the tight-binding inhibitor A70450 (a synthetic hexapeptide analogue) and with the general aspartic proteinase inhibitor pepstatin A (a microbial natural product) have been determined to 2.1 A and 3.0 A resolution, respectively. Although the protein structure retains the main features of a typical aspartic proteinase, it also shows some significant differences, due mainly to several sequence insertions and deletions (as revealed by homology modelling), that alter the shape of the binding cleft. There is also considerable variation in the C-terminal structural domain. CONCLUSIONS: The differences in side chains, and in the conformations adopted by the two inhibitors, particularly at their P4, P3 and P'2 positions (using standard notation for protease-inhibitor residues), allows the A70450 structure to complement, more accurately, that of the substrate-binding site of SAP2. Some differences in the binding clefts of other SAP isoenzymes may be deduced from the SAP2 structure.
Assuntos
Antifúngicos/química , Ácido Aspártico Endopeptidases/química , Candida albicans/enzimologia , Inibidores Enzimáticos/química , Proteínas Fúngicas/química , Isoenzimas/química , Modelos Moleculares , Pepstatinas/química , Piperazinas/química , Conformação Proteica , Sequência de Aminoácidos , Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/antagonistas & inibidores , Sítios de Ligação , Cristalografia por Raios X , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Proteínas Fúngicas/antagonistas & inibidores , Isoenzimas/antagonistas & inibidores , Substâncias Macromoleculares , Dados de Sequência Molecular , Pepstatinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
A crystal structure of a totally inactive insulin molecule has been determined. For this insulin molecule, the first without detectable activity to be characterized, the A and B-chains are linked by a peptide bond between A1 Gly and B29 Lys. The molecule has retained all its normal self-association properties and it can also accommodate the two different conformations designated T and R, as seen in 4Zn native pig insulin crystals. The hexamers of the crosslinked insulin molecule were crystallized using the 4Zn insulin recipe of Schlichtkrull. The structure has been crystallographically refined with data extending to 2 A using restrained least-square methods. Comparison of the B29-A1 peptide crosslink insulin and the 4Zn native insulin reveals close structural similarities with the native dimer. The analysis of the structure confirms the earlier hypothesis that insulin structures in crystals are not in an active conformation and that a separation of N-terminal A-chain and C-terminal B-chain is required for interaction with the insulin receptor.
Assuntos
Insulina/química , Sequência de Aminoácidos , Sítios de Ligação , Simulação por Computador , Reagentes de Ligações Cruzadas , Substâncias Macromoleculares , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Difração de Raios X/métodos , Zinco/metabolismoRESUMO
The prion protein PrP is a naturally occurring polypeptide that becomes transformed from a normal conformation to that of an aggregated form, characteristic of pathological states in fatal transmissible spongiform conditions such as Creutzfeld-Jacob Disease and Bovine Spongiform Encephalopathy. We report the crystal structure, at 2 A resolution, of residues 123-230 of the C-terminal globular domain of the ARQ allele of sheep prion protein (PrP). The asymmetric unit contains a single molecule whose secondary structure and overall organisation correspond to those structures of PrPs from various mammalian species determined by NMR. The globular domain shows a close association of helix-1, the C-terminal portion of helix-2 and the N-terminal portion of helix-3, bounded by the intramolecular disulphide bond, 179-214. The loop 164-177, between beta2 and helix-2 is relatively well structured compared to the human PrP NMR structure. Analysis of the sheep PrP structure identifies two possible loci for the initiation of beta-sheet mediated polymerisation. One of these comprises the beta-strand, residues 129-131 that forms an intra-molecular beta-sheet with residues 161-163. This strand is involved in lattice contacts about a crystal dyad to generate a four-stranded intermolecular beta-sheet between neighbouring molecules. The second locus involves the region 188-204, which modelling suggests is able to undergo a partial alpha-->beta switch within the monomer. These loci provide sites within the PrPc monomer that could readily give rise to early intermediate species on the pathway to the formation of aggregated PrPSc containing additional intermolecular beta-structure.
Assuntos
Príons/química , Animais , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Dimerização , Humanos , Modelos Moleculares , Doenças Priônicas/etiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , OvinosRESUMO
The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data. Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees. Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions. X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively. Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other. The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Ciclotrons , Modelos Químicos , Óperon/genética , Conformação Proteica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Triptofano/análogos & derivados , Triptofano/químicaRESUMO
We report the refined structure of a ternary complex of an allosterically activated lactate dehydrogenase, including the important active site loop. Eightfold non-crystallographic symmetry averaging was utilized to improve the density maps. Interactions between the protein and bound coenzyme and oxamate are described in relation to other studies using site-specific mutagenesis. Fructose 1,6-bisphosphate (FruP2) is bound to the enzyme across one of the 2-fold axes of the tetramer, with the two phosphate moieties interacting with two anion binding sites, one on each of two subunits, across this interface. However, because FruP2 binds at this special site, yet does not possess an internal 2-fold symmetry axis, the ligand is statistically disordered and binds to each site in two different orientations. Binding of FruP2 to the tetramer is signalled to the active site principally through two interactions with His188 and Arg173. His188 is connected to His195 (which binds the carbonyl group of the substrate) and Arg173 is connected to Arg171 (the residue that binds the carboxylate group of the substrate).
Assuntos
Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/ultraestrutura , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia , Análise Mutacional de DNA , Frutosefosfatos/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Dados de Sequência Molecular , NAD/metabolismo , Aceleradores de Partículas , Difração de Raios XRESUMO
The small, DNA-binding protein GerE regulates gene transcription in the terminally differentiated mother-cell compartment during late stages of sporulation in Bacillus subtilis. This versatile transcription factor shares sequence homology with the LuxR/FixJ/UhpA family of activators and modulates the expression of a number of genes, in particular those encoding the components of the coat that surrounds the mature spore. GerE orchestrates the final stages of coat deposition and maturation that lead to a spore with remarkable resistance properties but that must be responsive to low levels of germination signals. As this germination process is largely passive and can occur in the absence of de novo protein synthesis, the correct assembly of germination machinery, including germinant receptors and energy storage compounds, is crucial to the survival of the cell. The crystal structure of GerE has been solved at 2.05 A resolution using multi-wavelength anomalous dispersion techniques and reveals the nature of the GerE dimer. Each monomer comprises four alpha-helices, of which the central pair forms a helix-turn-helix DNA-binding motif. Implications for DNA-binding and the structural organisation of the LuxR/FixJ/UhpA family of transcription activator domains are discussed.
Assuntos
Bacillus subtilis/química , Proteínas de Bactérias/química , Fator sigma , Esporos Bacterianos/metabolismo , Fatores de Transcrição/química , Sequência de Aminoácidos , Bacillus subtilis/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Dimerização , Regulação Bacteriana da Expressão Gênica , Sequências Hélice-Volta-Hélice , Modelos Moleculares , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade Estática , Fatores de Transcrição/metabolismoRESUMO
Mycobacterium tuberculosis is the cause of tuberculosis in humans, a disease that affects over a one-third of the world's population. This slow-growing pathogen has only one ribosomal RNA operon, thus making its transcriptional apparatus a fundamentally interesting target for drug discovery. NusA binds to RNA polymerase and modulates several of the ribosomal RNA transcriptional processes. Here, we report the crystal structure of NusA, and reveal that the molecule consists of four domains. They are organised as two distinct entities. The N-terminal domain (residues 1 to 99) that resembles the B chain of the Rad50cd ATP binding cassette-ATPase (ABC-ATPase) and a C-terminal module (residues 108 to 329) consisting of a ribosomal S1 protein domain followed by two K homology domains. The S1 and KH domains are tightly integrated together to form an extensive RNA-binding structure, but are flexibly tethered to the N-terminal domain. The molecule's surfaces and architecture provide insights into RNA and polymerase interactions and the mechanism of pause site discrimination. They also allow us to rationalize certain termination-defective and cold shock-sensitive mutations in the nusA gene that have been studied in Escherichia coli.
Assuntos
Mycobacterium tuberculosis/química , Fatores de Alongamento de Peptídeos/química , Proteínas de Ligação a RNA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Proteínas de Escherichia coli , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Mutação Puntual/genética , Estrutura Terciária de Proteína , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Ribossômicas/química , Alinhamento de Sequência , Eletricidade Estática , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição , UltracentrifugaçãoRESUMO
The assembly of the insulin hexamer brings the six B13 glutamate side-chains at the centre into close proximity. Their mutual repulsion is unfavourable and zinc co-ordination to B10 histidine is necessary to stabilize the well known zinc-containing hexamers. Since B13 is always a carboxylic acid in all known sequences of hexamer forming insulins, it is likely to be important in the hormone's biology. The mutation of B13 Glu-->Gln leads to a stable zinc-free hexamer with somewhat reduced potency. The structures of the zinc-free B13 Gln hexamer and the 2Zn B13 insulin hexamer have been determined by X-ray analysis and refined with 2.5 A and 2.0 A diffraction data, respectively. Comparisons show that in 2Zn B13 Gln insulin, the hexamer structure (T6) is very like that of the native hormone. On the other hand, the zinc-free hexamer assumes a quaternary structure (T3/R3) seen in the native 4Zn insulin hexamer, and normally associated only with high chloride ion concentrations in the medium. The crystal structures show the B13 Gln side-chains only contact water in contrast to the B13 glutamate in 2Zn insulin. The solvation of the B13 Gln may be associated with this residue favouring helix at B1 to B8. The low potency of the B13 Gln insulin also suggests the residue influences the hormone's conformation.
Assuntos
Insulina/química , Conformação Proteica , Animais , Cristalização , Glutamatos , Ácido Glutâmico , Glutamina , Humanos , Insulina/metabolismo , Substâncias Macromoleculares , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura , Suínos , Difração de Raios X , Zinco/químicaRESUMO
Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilustrp operon is regulated by an attenuation mechanism similar to that which controls the trp operon in Bacillus subtilis. Secondary structure predictions indicate that the leader region of the trp mRNA is capable of folding into terminator and anti- terminator RNA structures. B. stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B. subtilis. The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution. Like the B. subtilis protein, B. stearothermophilus TRAP has 11 subunits arranged in a ring-like structure. The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B. stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B. subtilis protein, suggesting that neither of these properties is functionally important. However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way. As expected, the TRAP:RNA complex from B. stearothermophilus is significantly more thermostable than that from B. subtilis, with optimal binding occurring at 70 degrees C.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Geobacillus stearothermophilus/química , Óperon , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Geobacillus stearothermophilus/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Conformação Proteica , RNA Bacteriano , Sequências Reguladoras de Ácido Nucleico , Ribossomos , Homologia de Sequência de Aminoácidos , Triptofano/metabolismoRESUMO
The X-ray structure of tryptophanase (Tnase) reveals the interactions responsible for binding of the pyridoxal 5'-phosphate (PLP) and atomic details of the K+ binding site essential for catalysis. The structure of holo Tnase from Proteus vulgaris (space group P2(1)2(1)2(1) with a = 115.0 A, b = 118.2 A, c = 153.7 A) has been determined at 2.1 A resolution by molecular replacement using tyrosine phenol-lyase (TPL) coordinates. The final model of Tnase, refined to an R-factor of 18.7%, (Rfree = 22.8%) suggests that the PLP-enzyme from observed in the structure is a ketoenamine. PLP is bound in a cleft formed by both the small and large domains of one subunit and the large domain of the adjacent subunit in the so-called "catalytic" dimer. The K+ cations are located on the interface of the subunits in the dimer. The structure of the catalytic dimer and mode of PLP binding in Tnase resemble those found in aspartate amino-transferase, TPL, omega-amino acid pyruvate aminotransferase, dialkylglycine decarboxylase (DGD), cystathionine beta-lyase and ornithine decarboxylase. No structural similarity has been detected between Tnase and the beta 2 dimer of tryptophan synthase which catalyses the same beta-replacement reaction. The single monovalent cation binding site of Tnase is similar to that of TPL, but differs from either of those in DGD.