RESUMEN
Stress and genotype elicit changes in impulse control in a range of species that are attributable to adaptations in both the central and peripheral nervous system. We examined aspects of this mechanism in the horse by assessing the effect of a dopamine receptor genotype (DRD4) and central dopaminergic tone (measured via spontaneous blink rate [SBR] and behavioral initiation rate [BIR]), on measures of impulsivity, compulsivity (3-choice serial reaction time task) and sympathetic/ parasympathetic system balance (heart rate variability [HRV]). Genotype did not have a significant effect on any of the parameters measured. SBR but not BIR correlated significantly with levels of impulsivity. There was no clear association of HRV parameters with either measures of central dopaminergic activity or impulsivity/compulsivity. Overall, some elements of the data suggest that the horse may be a useful animal model for assessing the genetic and environmental factors that lead to the physiological and behavioral phenotype of human addiction, particularly when considering the relationship between central dopaminergic tone and impulsivity.
Asunto(s)
Conducta Adictiva , Conducta Impulsiva , Animales , Dopamina/farmacología , Genotipo , Caballos , Humanos , Receptores Dopaminérgicos , Especificidad de la EspecieRESUMEN
Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease affecting the joints. A heterogeneous response to available therapies demonstrates the need to identify those patients likely to benefit from a particular therapy. Our objective was to identify genetic factors associated with response to tocilizumab, a humanized monoclonal antibody targeting the interleukin (IL)-6 receptor, recently approved for treating RA. We report the first genome-wide association study on the response to tocilizumab in 1683 subjects with RA from six clinical studies. Putative associations were identified with eight loci, previously unrecognized as linked to the IL-6 pathway or associated with RA risk. This study suggests that it is unlikely that a major genetic determinant of response exists, and it illustrates the complexity of performing genome-wide association scans in clinical trials.
Asunto(s)
Anticuerpos Monoclonales Humanizados/administración & dosificación , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Interleucina-6/genética , Adulto , Antirreumáticos/administración & dosificación , Artritis Reumatoide/patología , Ensayos Clínicos Fase III como Asunto , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Interleucina-6/metabolismo , Masculino , Metotrexato/administración & dosificación , Persona de Mediana Edad , Polimorfismo de Nucleótido SimpleRESUMEN
The recent crystallographic characterization of NrfAs from Sulfurospirillum deleyianum, Wolinella succinogenes, Escherichia coli and Desulfovibrio desulfuricans allows structurally conserved regions to be identified. Comparison of nitrite and sulphite reductase activities from different bacteria shows that the relative activities vary according to organism. By comparison of both amino acid sequences and structures, differences can be identified in the monomer-monomer interface and the active-site channel; these differences could be responsible for the observed variance in substrate activity and indicate that subtle changes in the NrfA structure may optimize the enzyme for different roles.
Asunto(s)
Citocromos a1 , Citocromos c1 , Desulfovibrio desulfuricans/enzimología , Epsilonproteobacteria/enzimología , Escherichia coli/enzimología , Nitrato Reductasas , Conformación Proteica , Wolinella/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Grupo Citocromo c/química , Grupo Citocromo c/genética , Grupo Citocromo c/metabolismo , Citocromos a1/química , Citocromos a1/genética , Citocromos a1/metabolismo , Citocromos c1/química , Citocromos c1/genética , Citocromos c1/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Nitrato Reductasas/química , Nitrato Reductasas/genética , Nitrato Reductasas/metabolismo , Alineación de SecuenciaRESUMEN
Stress-induced changes in mesoaccumbens dopamine neurophysiology have been associated with the development of stereotypic behaviour in in-bred strains of laboratory rodents. This experiment evaluated whether similar changes are associated with environmentally-induced stereotypic behaviour in a higher-vertebrate species, the horse. D1- and D2-like dopamine receptor densities (B(max)) and dissociation constants (K(d)) were measured in control (n=9) and stereotypy (n=9) horses in the nucleus accumbens, caudate nucleus, putamen, substantia nigra and ventral tegmentum brain regions. Results revealed that stereotypy horses had significantly higher (P<0.05) dopamine D1 and D2 receptor densities (B(max)) in the nucleus accumbens compared to non-stereotypy controls. D1 receptor densities (B(max)) and D2 receptor affinity (K(d)) were also significantly lower in the caudate nucleus brain region of stereotypy horses (P<0.05). No other significant results were observed. These results demonstrate that stereotypy horses have increased activity within the mesoaccumbens dopamine pathway and, thus, that the development of environmentally-induced stereotypy may be associated with changes in motivational systems within the animal.
Asunto(s)
Conducta Animal/fisiología , Cuerpo Estriado/fisiología , Dopamina/fisiología , Núcleo Accumbens/fisiología , Conducta Estereotipada/fisiología , Sustancia Negra/fisiología , Animales , Femenino , Caballos , Masculino , Vías Nerviosas/fisiología , Receptores Dopaminérgicos/fisiologíaRESUMEN
The recent structural characterization of the NrfA from Escherichia coli provides a framework to rationalize the spectroscopic and functional properties of this enzyme. Analyses by EPR and magnetic CD spectroscopies have been complemented by protein-film voltammetry and these are discussed in relation to the essential structural features of the enzyme.
Asunto(s)
Citocromos a1/química , Citocromos c1/química , Escherichia coli/enzimología , Nitrato Reductasas/química , Dicroismo Circular , Espectroscopía de Resonancia por Spin del Electrón , Modelos Moleculares , Conformación ProteicaRESUMEN
Rhizobium leguminosarum fur mutants were unaffected in Fe-dependent regulation of several operons that specify different Fe uptake systems, yet cloned R. leguminosarum fur partially corrected an Escherichia coli fur mutant and R. leguminosarum Fur protein bound to canonical fur boxes. The lack of a phenotype in fur mutants is not due to functional redundancy with Irr, another member of the Fur superfamily found in the rhizobia, since irr fur double mutants are also unaffected in Fe-responsive regulation of several operons involved in Fe uptake. Neither Irr nor Fur is needed for symbiotic N(2) fixation on peas. As in Bradyrhizobium japonicum, irr mutants accumulated protoporphyrin IX. R. leguminosarum irr is not regulated by Fur and its Irr protein lacks the motif needed for haem-dependent post-translational modification that occurs in B. japonicum Irr. The similarities and differences in the Fur superfamily in the rhizobia and other Gram-negative bacteria are discussed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Hierro/metabolismo , Mutación , Proteínas Represoras/metabolismo , Rhizobium leguminosarum/metabolismo , Aldehído Oxidorreductasas/genética , Aldehído Oxidorreductasas/metabolismo , Proteínas Bacterianas/genética , Fijación del Nitrógeno , Pisum sativum/microbiología , Rhizobium leguminosarum/genética , Rhizobium leguminosarum/crecimiento & desarrollo , Simbiosis , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
The X-ray crystal structure of the apo-form of the Fur protein from Rhizobium leguminosarum has been solved at 2.7 A resolution. Small-angle X-ray scattering was used to give information on the solution conformation of the protein. The Fur homodimer folds into two domains. The N-terminal domain is formed from the packing of two helix-turn-helix motifs while the C-terminal domain appears primarily to stabilize the dimeric state of the protein.
Asunto(s)
Proteínas Bacterianas/química , Hierro/metabolismo , Proteínas Represoras/química , Rhizobium leguminosarum/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Metaloproteínas/química , Modelos Moleculares , Conformación ProteicaRESUMEN
A key component of the oxidative biogeochemical sulphur cycle involves the utilization by bacteria of reduced inorganic sulphur compounds as electron donors to photosynthetic or respiratory electron transport chains. The SoxAX protein of the photosynthetic bacterium Rhodovulum sulfidophilum is a heterodimeric c-type cytochrome that is involved in the oxidation of thiosulphate and sulphide. The recently solved crystal structure of the SoxAX complex represents the first structurally characterized example of a productive electron transfer complex between haemoproteins where both partners adopt the c-type cytochrome fold. The packing of c-type cytochrome domains both within SoxA and at the interface between the subunits of the complex has been compared with other examples and found to be unique.
Asunto(s)
Proteínas Bacterianas , Grupo Citocromo c/química , Oxidorreductasas/química , Rhodospirillaceae/enzimología , Sitios de Unión , Cristalografía por Rayos X , Hemo/metabolismo , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Subunidades de Proteína/químicaRESUMEN
Colicin endonucleases and the H-N-H family of homing enzymes share a common active site structural motif that has similarities to the active sites of a variety of other nucleases such as the non-specific endonuclease from Serratia and the sequence-specific His-Cys box homing enzyme I-PpoI. In contrast to these latter enzymes, however, it remains unclear how H-N-H enzymes cleave nucleic acid substrates. Here, we show that the H-N-H enzyme from colicin E9 (the E9 DNase) shares many of the same basic enzymological characteristics as sequence-specific H-N-H enzymes including a dependence for high concentrations of Mg2+ or Ca2+ with double-stranded substrates, a high pH optimum (pH 8-9) and inhibition by monovalent cations. We also show that this seemingly non-specific enzyme preferentially nicks double-stranded DNA at thymine bases producing 3'-hydroxy and 5'-phosphate termini, and that the enzyme does not cleave small substrates, such as dinucleotides or nucleotide analogues, which has implications for its mode of inhibition in bacteria by immunity proteins. The E9 DNase will also bind single-stranded DNA above a certain length and in a sequence-independent manner, with transition metals such as Ni2+ optimal for cleavage but Mg2+ a poor cofactor. Ironically, the H-N-H motif of the E9 DNase although resembling the zinc binding site of a metalloenzyme does not support zinc-mediated hydrolysis of any DNA substrate. Finally, we demonstrate that the E9 DNase also degrades RNA in the absence of metal ions. In the context of current structural information, our data show that the H-N-H motif is an adaptable catalytic centre able to hydrolyse nucleic acid by different mechanisms depending on the substrate and metal ion regime.
Asunto(s)
Colicinas/metabolismo , ADN/metabolismo , Endonucleasas/metabolismo , ARN/metabolismo , Serratia marcescens/enzimología , Secuencias de Aminoácidos , Naftalenosulfonatos de Anilina , Secuencia de Bases , Sitios de Unión , Calorimetría , Cationes Bivalentes/metabolismo , Coenzimas/metabolismo , Colicinas/química , ADN/química , ADN/genética , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Endonucleasas/química , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Hidrólisis , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Oligonucleótidos/química , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Conformación Proteica , ARN/química , ARN/genética , Espectrometría de Fluorescencia , Especificidad por Sustrato , TermodinámicaRESUMEN
The complex between the ribonuclease domain of the ribosome-inactivating colicin E3 and its protein inhibitor, the cognate immunity Im3, has been crystallized and preliminary X-ray characterization has been performed. Single crystals of the 1:1 complex were grown from hanging-drop vapour-diffusion experiments using 2-propanol as a precipitant. The space group is P3(1)21 or P3(2)21, with unit-cell parameters a = b = 93.7, c = 76.2 A. When cryocooled, these crystals diffract to a resolution of 2.4 A. A search for suitable conventional heavy-atom derivatives was unsuccessful and so Im3 mutants containing engineered cysteine or methionine residues have been produced for mercury soaks and selenomethionine-labelling experiments, respectively.
Asunto(s)
Proteínas Bacterianas/química , Colicinas/química , Proteínas de Escherichia coli , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Sitios de Unión , Colicinas/genética , Cristalización , Cristalografía por Rayos X , Escherichia coli/química , Mercurio/química , Metionina/química , Metionina/genética , Mutagénesis Sitio-Dirigida , Conformación Proteica , Ribonucleasas/química , Selenometionina/química , Serina/química , Serina/genéticaRESUMEN
The mechanism by which E colicins recognize and then bind to BtuB receptors in the outer membrane of Escherichia coli cells is a poorly understood first step in the process that results in cell killing. Using N- and C-terminal deletions of the N-terminal 448 residues of colicin E9, we demonstrated that the smallest polypeptide encoded by one of these constructs that retained receptor-binding activity consisted of residues 343-418. The results of the in vivo receptor-binding assay were supported by an alternative competition assay that we developed using a fusion protein consisting of residues 1-497 of colicin E9 fused to the green fluorescent protein as a fluorescent probe of binding to BtuB in E. coli cells. Using this improved assay, we demonstrated competitive inhibition of the binding of the fluorescent fusion protein by the minimal receptor-binding domain of colicin E9 and by vitamin B12. Mutations located in the minimum R domain that abolished or reduced the biological activity of colicin E9 similarly affected the competitive binding of the mutant colicin protein to BtuB. The sequence of the 76-residue R domain in colicin E9 is identical to that found in colicin E3, an RNase type E colicin. Comparative sequence analysis of colicin E3 and cloacin DF13, which is also an RNase-type colicin but uses the IutA receptor to bind to E. coli cells, revealed significant sequence homology throughout the two proteins, with the exception of a region of 92 residues that included the minimum R domain. We constructed two chimeras between cloacin DF13 and colicin E9 in which (i) the DNase domain of colicin E9 was fused onto the T+R domains of cloacin DF13; and (ii) the R domain and DNase domain of colicin E9 were fused onto the T domain of cloacin DF13. The killing activities of these two chimeric colicins against indicator strains expressing BtuB or IutA receptors support the conclusion that the 76 residues of colicin E9 confer receptor specificity. The minimum receptor-binding domain polypeptide inhibited the growth of the vitamin B12-dependent E. coli 113/3 mutant cells, demonstrating that vitamin B12 and colicin E9 binding is mutually exclusive.
Asunto(s)
Colicinas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/crecimiento & desarrollo , Péptidos/química , Receptores de Péptidos/metabolismo , Vitamina B 12/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa , Unión Competitiva , Cloacina/metabolismo , Clonación Molecular , Colicinas/química , Colicinas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Transporte de Membrana , Datos de Secuencia Molecular , Mutagénesis , Péptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Vitamina B 12/químicaRESUMEN
The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the 24 kDa complex of uniformly 13C and 15N labeled Im9 bound to the unlabeled DNase domain have provided sufficient constraints for the solution structure of the bound Im9 to be determined. For the final ensemble of 20 structures, pairwise RMSDs for residues 3-84 were 0.76 +/- 0.14 A for the backbone atoms and 1.36 +/- 0.15 A for the heavy atoms. Representative solution structures of the free and bound Im9 are highly similar, with backbone and heavy atom RMSDs of 1.63 and 2.44 A, respectively, for residues 4-83, suggesting that binding does not cause a major conformational change in Im9. The NMR studies have also allowed the DNase contact surface on Im9 to be investigated through changes in backbone chemical shifts and NOEs between the two proteins determined from comparisons of 1H-1H-13C NOESY-HSQC spectra with and without 13C decoupling. The NMR-defined interface agrees well with that determined in a recent X-ray structure analysis with the major difference being that a surface loop of Im9, which is at the interface, has a different conformation in the solution and crystal structures. Tyr54, a key residue on the interface, is shown to exhibit NMR characteristics indicative of slow rotational flipping. A mechanistic description of the influence binding of Im9 has on the dynamic behavior of E9 DNase, which is known to exist in two slowly interchanging conformers in solution, is proposed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Colicinas , Desoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
BACKGROUND: The cytotoxicity of most ribonuclease E colicins towards Escherichia coli arises from their ability to specifically cleave between bases 1493 and 1494 of 16S ribosomal RNA. This activity is carried by the C-terminal domain of the colicin, an activity which if left unneutralised would lead to destruction of the producing cell. To combat this the host E. coli cell produces an inhibitor protein, the immunity protein, which forms a complex with the ribonuclease domain effectively suppressing its activity. RESULTS: We have solved the crystal structure of the cytotoxic domain of the ribonuclease colicin E3 in complex with its immunity protein, Im3. The structure of the ribonuclease domain, the first of its class, reveals a highly twisted central beta-sheet elaborated with a short N-terminal helix, the residues of which form a well-packed interface with the immunity protein. CONCLUSIONS: The structure of the ribonuclease domain of colicin E3 is novel and forms an interface with its inhibitor which is significantly different in character to that reported for the DNase colicin complexes with their immunity proteins. The structure also gives insight into the mode of action of this class of enzymatic colicins by allowing the identification of potentially catalytic residues. This in turn reveals that the inhibitor does not bind at the active site but rather at an adjacent site, leaving the catalytic centre exposed in a fashion similar to that observed for the DNase colicins. Thus, E. coli appears to have evolved similar methods for ensuring efficient inhibition of the potentially destructive effects of the two classes of enzymatic colicins.
Asunto(s)
Colicinas/química , Colicinas/farmacología , Ribonucleasas/antagonistas & inhibidores , Ribonucleasas/química , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , ARN Ribosómico 16S/química , ARN Ribosómico 16S/metabolismo , Ribosomas/efectos de los fármacos , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein. Data for the Co2+-substituted and Ni2+-substituted proteins show that the metal ion is coordinated by three histidine residues; and the NMR characteristics of the Ni2+-substituted protein show that two of the histidines are coordinated through their N(epsilon2) atoms and one via its N(delta1). Furthermore, the NMR spectrum of the Ni2+-substituted protein is perturbed by the presence of phosphate, consistent with an X-ray structure showing that phosphate is coordinated to bound Ni2+, and by a change in pH, consistent with an ionisable group at the metal centre with a pKa of 7.9. Binding of an inhibitor protein to the DNase does not perturb the resonances of the metal site, suggesting there is no substantial conformation change of the DNase HNH motif on inhibitor binding. 1H-15N NMR data for the Zn2+-substituted DNase show that this protein, like the metal-free DNase, exists as two conformers with different 1H-15N correlation NMR spectra, and that the binding of Zn2+ does not significantly perturb the spectra, and hence structures, of these conformers beyond the HNH motif region.
Asunto(s)
Colicinas/química , Níquel/metabolismo , Dedos de Zinc , Zinc/metabolismo , Sitios de Unión , Colicinas/metabolismo , Desoxirribonucleasas/química , Desoxirribonucleasas/metabolismo , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética/métodos , Modelos Moleculares , Estructura Secundaria de ProteínaRESUMEN
The bacterial toxin colicin E9 is secreted by producing Escherichia coli cells with its 9.5 kDa inhibitor protein Im9 bound tightly to its 14.5 kDa C-terminal DNase domain. Double- and triple-resonance NMR spectra of the isolated DNase domain uniformly labeled with 13C/15N bound to unlabeled Im9 contain more signals than expected for a single DNase conformer, consistent with the bound DNase being present in more than one form. The presence of chemical exchange cross peaks in 750 MHz 15N-1H-15N HSQC-NOESY-HSQC spectra for backbone NH groups of Asp20, Lys21, Trp22, Leu23, Lys69, and Asn70 showed that the bound DNase was in dynamic exchange. The rate of exchange from the major to the minor form was determined to be 1.1 +/- 0.2 s(-1) at 298 K. Previous NMR studies have shown that the free DNase interchanges between two conformers with a forward rate constant of 1.61 +/- 0.11 s(-1) at 288 K, and that the bound Im9 is fixed in one conformation. The NMR studies of the bound DNase show that Im9 binds similarly to both conformers of the DNase and that the buried Trp22 is involved in the dynamic process. For the free DNase, all NH groups within a 9 A radius of any point of the Trp22 ring exhibit heterogeneity suggesting that a rearrangement of the position of this side chain is connected with the conformational interchange. The possible functional significance of this feature of the DNase is discussed.
Asunto(s)
Desoxirribonucleasas/antagonistas & inhibidores , Desoxirribonucleasas/química , Inhibidores Enzimáticos/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Conformación ProteicaRESUMEN
A leucine-rich repeat plant protein involved in resistance to pathogens, a polygalacturonase-inhibiting protein (PGIP-1) from Phaseolus vulgaris, has been crystallized and preliminary X-ray characterization has been performed. The protein contains ten repeats of a short (24 amino-acid) leucine-rich repeat motif. Single crystals of the protein were grown from vapour-diffusion experiments using PEG 2K monomethylether as precipitant; these crystals diffract to at least 2.3 A resolution. The space group is P2(1), with two molecules of PGIP-1 in the asymmetric unit; the crystals contain approximately 38% solvent.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Fabaceae/química , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Plantas Medicinales , Poligalacturonasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Fabaceae/genética , Solanum lycopersicum/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
BACKGROUND: E colicin proteins have three functional domains, each of which is implicated in one of the stages of killing Escherichia coli cells: receptor binding, translocation and cytotoxicity. The central (R) domain is responsible for receptor-binding activity whereas the N-terminal (T) domain mediates translocation, the process by which the C-terminal cytotoxic domain is transported from the receptor to the site of its cytotoxicity. The translocation of enzymatic E colicins like colicin E9 is dependent upon TolB but the details of the process are not known. RESULTS: We have demonstrated a protein-protein interaction between the T domain of colicin E9 and TolB, an essential component of the tol-dependent translocation system in E. coli, using the yeast two-hybrid system. The crystal structure of TolB, a procaryotic tryptophan-aspartate (WD) repeat protein, reveals an N-terminal alpha + beta domain based on a five-stranded mixed beta sheet and a C-terminal six-bladed beta-propeller domain. CONCLUSIONS: The results suggest that the TolB-box residues of the T domain of colicin E9 interact with the beta-propeller domain of TolB. The protein-protein interactions of other beta-propeller-containing proteins, the yeast yPrp4 protein and G proteins, are mediated by the loops or outer sheets of the propeller blades. The determination of the three-dimensional structure of the T domain-TolB complex and the isolation of mutations in TolB that abolish the interaction with the T domain will reveal fine details of the protein-protein interaction of TolB and the T domain of E colicins.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Colicinas/química , Colicinas/metabolismo , Proteínas de Escherichia coli , Proteínas Periplasmáticas , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Colicinas/genética , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Estructura Terciaria de Proteína , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Electricidad Estática , Técnicas del Sistema de Dos HíbridosRESUMEN
Homing endonucleases are classified into four families based on active site sequence motifs. Through structural comparisons we have found structural similarities between the endonuclease domain of colicin E9, an H-N-H motif-containing enzyme, and both the non-specific nuclease from Serratia and I-PpoI, a His-Cys box-containing homing endonuclease. Our comparison identifies conservation at the heart of all three enzyme active sites and so argues for a re-classification of H-N-H and His-Cys box homing endonucleases as a single family. We suggest the 'betabetaalpha-Me family' of homing enzymes to reflect the three elements of secondary structure and the metal ion that define the motif.
Asunto(s)
Colicinas/química , Endodesoxirribonucleasas/química , Endonucleasas/química , Endonucleasas/clasificación , Aminoácidos/química , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Sitios de Unión , Colicinas/genética , Secuencia Conservada , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de ProteínaRESUMEN
Fumarate reductases and succinate dehydrogenases play central roles in the metabolism of eukaryotic and prokaryotic cells. A recent medium resolution structure of the Escherichia coli fumarate reductase (Frd) has revealed the overall organization of the membrane-bound complex. Here we present the first high resolution X-ray crystal structure of a water-soluble bacterial fumarate reductase in an open conformation. This structure reveals a mobile domain that modulates substrate access to the active site and provides new insights into the mechanism of this widespread and important family of FAD-containing respiratory proteins.
Asunto(s)
Grupo Citocromo c/química , Grupo Citocromo c/metabolismo , Shewanella/enzimología , Succinato Deshidrogenasa/química , Succinato Deshidrogenasa/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Dimerización , Flavinas/metabolismo , Fumaratos/metabolismo , Hemo/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Movimiento (Física) , Oxidación-Reducción , Conformación Proteica , Solubilidad , Electricidad Estática , Relación Estructura-ActividadRESUMEN
The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn(2+) or Ni(2+)) the role of which is unknown. In the present work we find that neither Zn(2+) nor Ni(2+) is required for DNase activity, which instead requires Mg(2+) ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn(2+), Ni(2+), or Co(2+), the thermodynamic stability of the domain increased substantially, and that the equilibrium dissociation constant for Zn(2+) was less than or equal to nanomolar, while that for Co(2+) and Ni (2+) was micromolar. Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.