RESUMEN
Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 A resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 A resolution and in a complex with NAD+ at 2.3 A resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.
Asunto(s)
Reductasas del Citocromo/química , Secuencia de Aminoácidos , Animales , Cristalización , Cristalografía por Rayos X , Reductasas del Citocromo/genética , Citocromo-B(5) Reductasa , Humanos , Lisina/genética , Metahemoglobinemia/enzimología , Metahemoglobinemia/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NAD/química , Conformación Proteica , Ratas , Homología de Secuencia de Aminoácido , PorcinosRESUMEN
Serum transferrin is the major iron transport protein in humans. Its function depends on its ability to bind iron with very high affinity, yet to release this bound iron at the lower intracellular pH. Possible explanations for the release of iron from transferrin at low pH include protonation of a histidine ligand and the existence of a pH-sensitive "trigger" involving a hydrogen-bonded pair of lysines in the N-lobe of transferrin. We have determined the crystal structure of the His249Glu mutant of the N-lobe half-molecule of human transferrin and compared its iron-binding properties with those of the wild-type protein and other mutants. The crystal structure, determined at 2.4 A resolution (R-factor 19.8%, R(free) 29.4%), shows that Glu 249 is directly bound to iron, in place of the His ligand, and that a local movement of Lys 296 has broken the dilysine interaction. Despite the loss of this potentially pH-sensitive interaction, the H249E mutant is only slightly more acid-stable than wild-type and releases iron slightly faster. We conclude that the loss of the dilysine interaction does make the protein more acid stable but that this is counterbalanced by the replacement of a neutral ligand (His) by a negatively charged one (Glu), thus disrupting the electroneutrality of the binding site.
Asunto(s)
Dipéptidos/metabolismo , Ácido Glutámico/genética , Histidina/genética , Hierro/metabolismo , Mutagénesis Sitio-Dirigida , Transferrina/genética , Sitios de Unión/genética , Cristalografía por Rayos X , Dipéptidos/química , Ácido Glutámico/metabolismo , Histidina/metabolismo , Humanos , Unión Proteica/genética , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transferrina/química , Transferrina/metabolismoRESUMEN
Binding of virus particles to specific host cell surface receptors is known to be an obligatory step in infection even though the molecular basis for these interactions is not well characterized. The crystal structure of the adenovirus fiber knob domain in complex with domain I of its human cellular receptor, coxsackie and adenovirus receptor (CAR), is presented here. Surface-exposed loops on knob contact one face of CAR, forming a high-affinity complex. Topology mismatches between interacting surfaces create interfacial solvent-filled cavities and channels that may be targets for antiviral drug therapy. The structure identifies key determinants of binding specificity, which may suggest ways to modify the tropism of adenovirus-based gene therapy vectors.
Asunto(s)
Adenovirus Humanos/metabolismo , Proteínas de la Cápside , Cápside/química , Cápside/metabolismo , Receptores Virales/química , Receptores Virales/metabolismo , Adenovirus Humanos/química , Sustitución de Aminoácidos , Sitios de Unión , Proteína de la Membrana Similar al Receptor de Coxsackie y Adenovirus , Cristalización , Cristalografía por Rayos X , Enlace de Hidrógeno , Modelos Moleculares , Mutagénesis , Unión Proteica , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TermodinámicaRESUMEN
BACKGROUND: Arginase is a manganese-dependent enzyme that catalyzes the hydrolysis of L-arginine to L-ornithine and urea. In ureotelic animals arginase is the final enzyme of the urea cycle, but in many species it has a wider role controlling the use of arginine for other metabolic purposes, including the production of creatine, polyamines, proline and nitric oxide. Arginase activity is regulated by various small molecules, including the product L-ornithine. The aim of these structural studies was to test aspects of the catalytic mechanism and to investigate the structural basis of arginase inhibition. RESULTS: We report here the crystal structures of arginase from Bacillus caldovelox at pH 5.6 and pH 8.5, and of binary complexes of the enzyme with L-arginine, L-ornithine and L-lysine at pH 8.5. The arginase monomer comprises a single compact alpha/beta domain that further associates into a hexameric quaternary structure. The binary complexes reveal a common mode of ligand binding, which places the substrate adjacent to the dimanganese centre. We also observe a conformational change that impacts on the active site and is coupled with the occupancy of an external site by guanidine or arginine. CONCLUSIONS: The structures reported here clarify aspects of the active site and indicate key features of the catalytic mechanism, including substrate coordination to one of the manganese ions and an orientational role for a neighboring histidine residue. Stereospecificity for L-amino acids is found to depend on their precise recognition at the active-site rim. Identification of a second arginine-binding site, remote from the active site, and associated conformational changes lead us to propose a regulatory role for this site in substrate hydrolysis.
Asunto(s)
Arginasa/química , Bacillus/enzimología , Proteínas Bacterianas/química , Conformación Proteica , Arginasa/antagonistas & inhibidores , Arginasa/metabolismo , Arginina/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Sitios de Unión , Unión Competitiva , Catálisis , Cristalografía por Rayos X , Activación Enzimática , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Lisina/metabolismo , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Familia de Multigenes , Ornitina/metabolismo , Unión Proteica , Proteínas Recombinantes de Fusión/química , Espectrofotometría Atómica , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.
Asunto(s)
Lactoglobulinas/química , Isoformas de Proteínas/química , Sustitución de Aminoácidos , Animales , Bovinos , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Modelos Moleculares , Conformación ProteicaRESUMEN
The ferric form of the N-lobe of human serum transferrin (Fe(III)-hTF/2N) has been expressed at high levels in Pichia pastoris. The Fe(III)-hTF/2N was crystallized in the space group P41212, and X-ray crystallography was used to solve the structure of the recombinant protein at 2.5 A resolution. This represents only the second P. pastoris-derived protein structure determined to date, and allows the comparison of the structures of recombinant Fe(III)-hTF/2N expressed in P. pastoris and mammalian cells with serum-derived transferrin. The polypeptide folding pattern is essentially identical in all of the three proteins. Mass spectroscopic analyses of P. pastoris- hTF/2N and proteolytically derived fragments revealed glycosylation of Ser-32 with a single hexose. This represents the first localization of an O-linked glycan in a P. pastoris-derived protein. Because of its distance from the iron-binding site, glycosylation of Ser-32 should not affect the iron-binding properties of hTF/2N expressed in P. pastoris, making this an excellent expression system for the production of hTF/2N.
Asunto(s)
Fragmentos de Péptidos/química , Pichia/genética , Pliegue de Proteína , Proteínas Recombinantes/química , Serina/metabolismo , Transferrina/química , Animales , Línea Celular , Cricetinae , Cristalización , Cristalografía por Rayos X , Compuestos Férricos/química , Glicosilación , Humanos , Riñón/citología , Espectrometría de Masas , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Serina/genética , Transferrina/genética , Transferrina/metabolismoRESUMEN
Serum transferrin binds ferric ions in the bloodstream and transports them to cells, where they are released in a process involving receptor-mediated endocytosis. Iron release is believed to be pH dependent and is coupled with a large conformational change. To help define the steps in iron release, we have determined the three-dimensional structure of the iron-free (apo) form of the recombinant N-lobe half-molecule of human serum transferrin (ApoTfN) by X-ray crystallography. Two crystal forms were obtained, form 1 with four molecules in the asymmetric unit and form 2 with two molecules in the asymmetric unit. The structures of both forms were determined by molecular replacement and were refined at 2.2 and 3.2 A resolution, respectively. Final R-factors were 0.203 (free R = 0. 292) for form 1 and 0.217 (free R = 0.312) for form 2. All six copies of the ApoTfN structure are essentially identical. Comparison with the holo form (FeTfN) shows that a large rigid-body domain movement of 63 degrees has occurred in ApoTfN, to give an open binding cleft. The extent of domain opening is the same as in the N-lobe of human lactoferrin, showing that it depends on internal constraints that are conserved in both proteins, and that it is unaffected by the presence or absence of the C-lobe. Although the conformational change is primarily a rigid-body motion, several local adjustments occur. In particular, two iron ligands, Asp 63 and His 249, change conformation to form salt bridges, with Lys 296 and Glu 83, respectively, in the binding cleft of the apo protein. Both salt bridges would have to break for iron coordination to occur. Most importantly, the structure, determined at a pH (5.3) that is close to the pH of physiological iron release, indicates that protonation of His 249 is a key step in iron release.
Asunto(s)
Fragmentos de Péptidos/química , Conformación Proteica , Transferrina/química , Apoproteínas/química , Apoproteínas/genética , Sitios de Unión , Simulación por Computador , Cristalización , Cristalografía por Rayos X , Compuestos Férricos/química , Humanos , Lactoferrina/química , Ligandos , Modelos Moleculares , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Transferrina/genética , Transferrina/metabolismoRESUMEN
The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.
Asunto(s)
Lactoglobulinas/química , Conformación Proteica , Animales , Bovinos , Simulación por Computador , Cristalografía por Rayos X , Electrones , Femenino , Concentración de Iones de Hidrógeno , Absorción Intestinal , Lactoglobulinas/fisiología , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox' has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coli using an inducible T7 RNA polymerase-based system. A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein.
Asunto(s)
Arginasa/química , Bacillus/enzimología , Secuencia de Aminoácidos , Arginasa/genética , Arginasa/metabolismo , Secuencia de Bases , Clonación Molecular , Cristalografía por Rayos X , ADN Bacteriano , Expresión Génica , Calor , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
The crystal structure of chicken annexin V has been solved by molecular replacement and refined at 2.25 A. The final R factor is 19.7% with good geometry. The chicken annexin V structure is very similar to the human annexin V structure, with four similar domains each containing five helices. The structure includes three calcium ions in domains I, II, and IV, each bound by the characteristic K-G-X-G-T-(38 residues)-D/E motif. In view of the structural similarity between human and chicken annexin V, we suggest that they have a common vital function which developed early in evolutionary history.