RESUMEN
Barstar, the natural inhibitor of barnase crystallizes in many different crystal forms under almost identical conditions. Although barstar is a monomeric protein, it crystallizes with four molecules in the asymmetric unit in two crystal forms, rhombohedral (space group R3; a = b = 118.0 angstrum; c = 75.5 angstrum) and tetragonal (space group P4; a = b = 105.1 angstrum; c = 36.0 angstrum), which exist simultaneously under identical crystallization conditions. The relation between the four molecules in the asymmetric unit of the crystals belonging to space group P4 can be interpreted in terms of a small distortion in the crystallographic symmetry of the higher symmetry space group P422.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Cristalización , Cristalografía por Rayos X , Estructura Molecular , Proteínas Recombinantes/biosíntesisRESUMEN
The GnRH associated prolactin inhibiting factor (GAP) reveals the signature sequence associated with the helix-loop-helix structural motif. A number of different peptide fragments of GAP were designed, synthesized and analysed by circular dichroism and by an in vivo assay for prolactin secretion inhibiting activity. Peptides corresponding to the two individual alpha-helices and a 44-residue peptide comprising the entire helix-loop-helix motif show significant helical propensity in circular dichroism spectra. However, a peptide corresponding to the loop sequence shows no helical propensity. Albeit, the peptide corresponding to helix-loop-helix motif was found to inhibit prolactin secretion and augment circulating levels of gonadotropins in the in vivo assay; other shorter peptides did not show such activity. The activity profile of the 44-residue peptide was biphasic and very similar to that of the recombinant GAP. Thus, the prolactin inhibiting activity of this factor is defined by its helix-loop-helix motif as in the case of the transcription factors of developmental genes. The structural features of a homology-based model of GAP in complex with E47, a ubiquitous HLH-type developmental gene regulator, are consistent with the structural requirements of the negative regulation of transcription by helix-loop-helix proteins.
Asunto(s)
Hormona Liberadora de Gonadotropina/química , Hormona Liberadora de Gonadotropina/farmacología , Secuencias Hélice-Asa-Hélice , Prolactina/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/farmacología , Factores de Transcripción , Secuencia de Aminoácidos , Dicroismo Circular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Dimerización , Hormona Folículo Estimulante/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hormona Luteinizante/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Transcripción Genética/genéticaRESUMEN
Bacterial flagellin has two domains: the polymerizing domain consisting of N- and C-terminal regions which are partly disordered in the monomeric state; and the central antigenic domain with compact globular structure. The polymerizing domain is highly conserved in flagellins from different species but the antigenic domain is diverse in sequence and size. Whereas the former has direct functional significance for bacterial motility, the latter has not been identified as having a specific function except for defining the distinct serotype of the bacterium. The sequence alignment of flagellin from S. paratyphi with proteins of known three-dimensional structure reveals significant homology of the central 265 residue stretch with the bacterial serine protease, subtilisin. This homology is evident also in the comparison of the predicted secondary structure of flagellin with the observed secondary structural features in subtilisin. The deletions/insertions arising due to optimal alignment of the two proteins occur on the surface loops in the structure. Thus, a domain of S. paratyphi flagellin and subtilisin appear to have similar structural folds.
Asunto(s)
Flagelina/química , Salmonella paratyphi A/química , Subtilisinas/química , Secuencia de Aminoácidos , Antígenos Bacterianos/química , Flagelina/inmunología , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Salmonella paratyphi A/inmunología , Homología de Secuencia de AminoácidoRESUMEN
Bromophenol red (BPR) binds to lysozyme and inhibits its activity against bacterial cell walls, but not against the polysaccharide component of peptidoglycan. The binding site of BPR in the enzyme has been characterised by X-ray analysis of the complex at 5.5A resolution. The new binding site, which is outside the cleft close to subsite F, is presumably involved in interactions with the peptide component of peptidoglycan, in the action of lysozyme against bacterial cell walls.
Asunto(s)
Muramidasa/metabolismo , Animales , Sitios de Unión , Pollos , Clara de Huevo , Micrococcus/enzimología , Modelos Moleculares , Fenolsulfonftaleína/análogos & derivados , Fenolsulfonftaleína/metabolismo , Unión Proteica , Conformación Proteica , Espectrofotometría , Difracción de Rayos XRESUMEN
We have earlier reported that overexpression of the gene encoding human hyaluronan-binding protein (HABP1) is functionally active, as it binds specifically with hyaluronan (HA). In this communication, we confirm the collapse of the filamentous and branched structure of HA by interaction with increasing concentrations of recombinant-HABP1 (rHABP1). HA is the reported ligand of rHABP1. Here, we show the affinity of rHABP1 towards D-mannosylated albumin (DMA) by overlay assay and purification using a DMA affinity column. Our data suggests that DMA is another ligand for HABP1. Furthermore, we have observed that DMA inhibits the binding of HA in a concentration-dependent manner, suggesting its multiligand affinity amongst carbohydrates. rHABP1 shows differential affinity towards HA and DMA which depends on pH and ionic strength. These data suggest that affinity of rHABP1 towards different ligands is regulated by the microenvironment.
Asunto(s)
Fibroblastos/metabolismo , Receptores de Hialuranos/metabolismo , Manosa/química , Manosa/metabolismo , Albúmina Sérica/química , Animales , Enzimas Inmovilizadas/metabolismo , Fibroblastos/química , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Ligandos , Proteínas Recombinantes/metabolismo , Sefarosa/química , Albúmina Sérica/metabolismoRESUMEN
L-Ornithine L-aspartate hemihydrate crystallizes in the space group C2 with a = 21.858(2), b = 4.718(1), c = 18.046(2) A and beta = 137.4(1) degrees. The crystal structure, solved by direct methods, has been refined to an R value of 0.041 for 1270 observed reflections. The conformation of the two amino acid molecules in the structure are somewhat different from those observed in other crystal structures which contain them. The crystal structure is stabilized by ionic interactions accompanied by hydrogen bonds. The unlike molecules aggregate into separate two-fold helices; each helix of one type is surrounded by, and is in hydrogen bonded contact with, four helices of the other type. The arrangement of the molecules in the structure is such that it can be described as consisting of alternating hydrophilic and hydrophobic regions. The hydrophilic regions contain hydrogen bonded loops, each made up of two amino groups and two carboxylate groups. The structure also provides the first example of a head-to-tail sequence involving two types of amino acids.
Asunto(s)
Dipéptidos , Cristalización , Enlace de Hidrógeno , Conformación Proteica , Temperatura , Difracción de Rayos XRESUMEN
The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in E. coli, was isolated as oligomers resembling the dissociated capsomeres derived from viral capsids. Image analysis of low-dose electron micrographs demonstrates that these VP1 oligomers are exclusively pentamers. The purified VP1 pentamers associated to form capsid-like assemblies and polymorphic aggregates at high ionic strength. The capsid-like assemblies were stabilized at low ionic strength by the addition of calcium. Self-assembly of the unmodified, recombinant DNA-generated VP1 implies that the posttranslational charge modifications of VP1 and the minor virion protein components, VP2 and VP3, are not essential for capsid formation. The nonequivalently related subunits of the penta- and hexavalent capsomeres therefore must spontaneously switch their bonding specificity during assembly.
Asunto(s)
Cápside/fisiología , Poliomavirus/ultraestructura , Clonación Molecular , Gráficos por Computador , Escherichia coli , Sustancias Macromoleculares , Microscopía Electrónica , Morfogénesis , Unión ProteicaRESUMEN
Specific functional properties of many proteins directing developmental responses via transcriptional regulation are orchestrated by their characteristic helix-loop-helix (HLH) structural motif. The entire HLH motif in all these proteins assumes a common conformation irrespective of their individual biological effects. The motif controls the affinity of HLH proteins for homo- or heterodimerization, permitting mixing and matching of regulatory factors, and thereby expanding the functional repertoire. Systematic analysis of molecular contacts at the dimer interface using the models built for the functional dimers combined with the pattern of conserved/nonconserved residues within different categories of HLH proteins helped in understanding the differential role played by different residues at the dimer interface for expressing corresponding functions. The residues associated with the self and partner interactions were identified, and the signature residues contributing toward dimeric stability and functional specificity were defined. It is evident that most of the residues involved in self interactions are common among all the HLH proteins. However, while certain residues involved in partner interactions are common among all the HLH proteins, certain others are common within a category, and still others vary widely defining specificity signature at different levels.
Asunto(s)
Proteínas de Unión al ADN/química , Secuencias Hélice-Asa-Hélice/fisiología , Factores de Transcripción/genética , Secuencia de Aminoácidos , Sitios de Unión , Secuencia Conservada , Genes Reguladores , Modelos Estructurales , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Nonequivalent bonding of identical protein subunits occurs in the polyomavirus capsid were identical pentameric capsomeres occupy both hexavalent and pentavalent positions in the icosahedral surface lattice. The polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, has been isolated as capsomeres that self-assemble into capsid-like structures in vitro. The ability to switch bonding specificity in different symmetry environments therefore must be intrinsic to the VP1 molecule. In vitro self-assembly provides an assay for VP1 mutations affecting capsomere and capsid formation. We report here that a directed mutation in the VP1 expression vector, leading to a protein truncated at the carboxy terminus, results in a mutant VP1 that forms capsomeres, but not capsids, in the in vitro assembly assay. The carboxy terminus of VP1 therefore appears to be involved in the specific bonding responsible for the non-equivalent association of capsomeres.
Asunto(s)
Cápside/genética , Mutación , Poliomavirus/genética , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Sustancias Macromoleculares , Microscopía ElectrónicaRESUMEN
Polyomavirus major capsid protein VP1, purified after expression of the recombinant gene in Escherichia coli, forms stable pentamers in low-ionic strength, neutral, or alkaline solutions. Electron microscopy showed that the pentamers, which correspond to viral capsomeres, can be self-assembled into a variety of polymorphic aggregates by lowering the pH, adding calcium, or raising the ionic strength. Some of the aggregates resembled the 500-A-diameter virus capsid, whereas other considerably larger or smaller capsids were also produced. The particular structures formed on transition to an environment favoring assembly depended on the pathway of the solvent changes as well as on the final conditions. Mass measurements from cryoelectron micrographs and image analysis of negatively stained specimens established that a distinctive 320-A-diameter particle consists of 24 close-packed pentamers arranged with octahedral symmetry. Comparison of this unexpected octahedral assembly with a 12-capsomere icosahedral aggregate and the 72-capsomere icosahedral virus capsid by computer graphics methods indicates that similar connections are made among trimers of pentamers in these shells of different size. The polymorphism in the assembly of VP1 pentamers can be related to the switching in bonding specificity required to build the virus capsid.
Asunto(s)
Proteínas de la Cápside , Cápside/ultraestructura , Poliomavirus/genética , Cápside/genética , Escherichia coli/genética , Genes Virales , Microscopía Electrónica , Modelos Moleculares , Polimorfismo Genético , Proteínas Recombinantes/ultraestructura , Proteínas Estructurales Virales/genéticaRESUMEN
The structures of concanavalin A (ConA) in complex with two carbohydrate-mimicking peptides, 10-mer (MYWYPYASGS) and 15-mer (RVWYPYGSYLTASGS) have been determined at 2.75 A resolution. In both crystal structures four independent peptide molecules bind to each of the crystallographically independent subunits of ConA tetramer. The peptides exhibit small but significant variability in conformations and interactions while binding to ConA. The crystal structure of another similar peptide, 12-mer (DVFYPYPYASGS), in complex with ConA has been determined (Jain, D., K. J. Kaur, B. Sundaravadivel, and D. M. Salunke. 2000. Structural and functional consequences of peptide-carbohydrate mimicry. J. Biol. Chem. 275:16098-16102). Comparison of the three complexes shows that the peptides bind to ConA at a common binding site, using different contacting residues and interactions depending on their sequence and the local environment at the binding site. The binding is also optimized by corresponding plasticity of the peptide binding site on ConA. The diversity in conformation and interactions observed here are in agreement with the structural leeway concerning plasticity of specific molecular recognition in biological processes. The adaptability of peptide-ConA interactions may also be correlated with the carbohydrate-mimicking property of these peptides.
Asunto(s)
Concanavalina A/química , Concanavalina A/metabolismo , Cristalografía por Rayos X , Péptidos/química , Péptidos/metabolismo , Modelos Moleculares , Péptidos/síntesis química , Unión Proteica , Conformación Proteica , Electricidad Estática , Especificidad por SustratoRESUMEN
Functionally important glycosylation has been identified in the antigen binding domain of an anti-GnRH monoclonal antibody. Presence of mannose and sialic acid residues is revealed from con A immunoblots and positive staining with a sialic acid detection kit, respectively. Desialylation of the antibody reduces GnRH binding, suggesting the role of terminal sialic acid residues in modulating antigen binding. The crystal structure of the Fab fragment shows electron density adjacent to the antigen binding site which may be attributed to the covalently attached carbohydrate moiety. Thus, the presence of sialic acid containing mannose-rich carbohydrate moiety near the antigen binding site of a monoclonal antibody Fab fragment is relevant for defining antibody specificity.
Asunto(s)
Anticuerpos Monoclonales/química , Hormona Liberadora de Gonadotropina/inmunología , Animales , Anticuerpos Monoclonales/metabolismo , Diversidad de Anticuerpos , Sitios de Unión de Anticuerpos , Carbohidratos/análisis , Concanavalina A , Cristalografía por Rayos X , Glicosilación , Immunoblotting , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/química , Región Variable de Inmunoglobulina/metabolismo , Manosa/química , Ratones , Modelos Moleculares , Ácido N-Acetilneuramínico/química , Conformación ProteicaRESUMEN
The shared surface topology of two chemically dissimilar but functionally equivalent molecular structures has been analyzed. A carbohydrate moiety (alpha-D-mannopyranoside) and a peptide molecule (DVFYPYPYASGS) bind to concanavalin A at a common binding site. The cross-reactivity of the polyclonal antibodies (pAbs) was used for understanding the topological relationship between these two independent ligands. The anti-alpha-D-mannopyranoside pAbs recognized various peptide ligands of concanavalin A, and the anti-DVFYPYPYASGS pAbs recognized the carbohydrate ligands, providing direct evidence of molecular mimicry. On the basis of differential binding of various rationally designed peptide analogs to the anti-alpha-D-mannopyranoside pAbs, it was possible to identify different peptide residues critical for the mimicry. The comparison of circular dichroism profiles of the designed analogs suggests that the carbohydrate mimicking conformation of the peptide ligand incorporates a polyproline type II structural fold. The concanavalin A binding activity of these analogs was found to have a direct correlation with the topological relationship between peptide and carbohydrate ligands.
Asunto(s)
Manosa/metabolismo , Oligopéptidos/metabolismo , Péptidos/metabolismo , Animales , Anticuerpos , Unión Competitiva , Conformación de Carbohidratos , Concanavalina A/metabolismo , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Ratones , Ratones Endogámicos BALB C , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The structural basis of affinity enhancement was addressed by analyzing the interactions between concanavalin A and the carbohydrate-mimicking peptide ligands. Based on the crystal structures of concanavalin A in complex with these peptides [Jain, D., Kaur, K. J., Sundaravadivel, B., and Salunke, D. M. (2000) J. Biol. Chem. 275, 16098-16102; Jain, D., Kaur, K. J., and Salunke, D. M. (2001) Biophys. J. 80, 2912-2921], a high-affinity analogue was designed. This analogue (acetyl-MYWYPY-amide) binds to the lectin with 32-fold enhanced affinity compared to the corresponding precursor peptides. The crystal structure of concanavalin A bound to the designed peptide has been determined. A peptide molecule binds to each of the crystallographically independent monomers of the tetrameric lectin. The four bound peptide molecules exhibit two major conformations both of which are extended. Unlike in the case of other concanavalin A binding peptides, the structural variations within different conformers of this analogue are marginal. It is apparent that the deletion of the structurally variable region of the larger peptides has led to an improved complementarity and increased buried surface area in the case of the designed peptide. The crystal structure also showed the formation of two backbone hydrogen bonds between the ligand and the ligate which were not present in the complexes of the precursor peptides. The observed structural features adequately explain the enhanced binding of the designed analogue.
Asunto(s)
Concanavalina A/metabolismo , Péptidos/metabolismo , Ligandos , Modelos Moleculares , Péptidos/síntesis química , Péptidos/química , Unión Proteica , Conformación ProteicaRESUMEN
Primary structural homology between the hormone binding site of the LH/CG receptor and the enzyme binding site of chymotrypsin inhibitor has been identified. This has led to the application of a knowledge-based approach of molecular modelling to describe the interaction of choriogonadotropin (CG) with the LH/CG receptor. A tertiary structural model for the mode of recognition between the hormone and the receptor has been proposed. As in other such processes at the molecular level, the recognition between CG and its receptor is mediated through non-covalent interactions. The specificity of recognition is achieved by complementarity in van der Waals surfaces, hydrogen bonding and non-polar associations. The model shows nine hydrogen bonds between the hormone and the receptor involving polar side chains as well as backbone amine and carbonyl groups. A hydrophobic cluster involving side chain groups at the interface is also important in stabilization of the intermolecular interactions.
Asunto(s)
Gonadotropina Coriónica/metabolismo , Simulación por Computador , Modelos Moleculares , Receptores de HL/metabolismo , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Secuencia de Aminoácidos , Quimotripsina/antagonistas & inhibidores , Quimotripsina/química , Humanos , Enlace de Hidrógeno , Datos de Secuencia Molecular , Unión Proteica , Estructura Terciaria de Proteína , Receptores de HL/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/química , Inhibidores de Serina Proteinasa/químicaRESUMEN
The functional consequences of peptide-carbohydrate mimicry were analyzed on the basis of the crystal structure of concanavalin A (ConA) in complex with a carbohydrate-mimicking peptide, DVFYPYPYASGS. The peptide binds to the non-crystallographically related monomers of two independent dimers of ConA in two different modes, in slightly different conformations, demonstrating structural adaptability in ConA-peptide recognition. In one mode, the peptide has maximum interactions with ConA, and in the other, it shows relatively fewer contacts within this site but significant contacts with the symmetry-related subunit. Neither of the peptide binding sites overlaps with the structurally characterized mannose and trimannose binding sites on ConA. Despite this, the functional mimicry between the peptide and carbohydrate ligands was evident. The peptide-inhibited ConA induced T cell proliferation in a dose-dependent manner. The effect of the designed analogs of the peptide on ConA-induced T cell proliferation and their recognition by the antibody response against alpha-d-mannopyranoside indicate a role for aromatic residues in functional mimicry. Although the functional mimicry was observed between the peptide and carbohydrate moieties, the crystal structure of the ConA-peptide complex revealed that the two peptide binding sites are independent of the methyl alpha-d-mannopyranoside binding site.
Asunto(s)
Concanavalina A/química , Péptidos/química , Sitios de Unión , División Celular/efectos de los fármacos , Cristalografía por Rayos X , Dimerización , Enlace de Hidrógeno , Manósidos/inmunología , Modelos Moleculares , Péptidos/farmacología , Unión Proteica , Conformación Proteica , Linfocitos TRESUMEN
The three-dimensional structure of rat pancreatic RNase A expressed in Escherichia coli was determined. The backbone conformations of certain critical loops are significantly different in this enzyme compared to its bovine counterpart. However, the core structure of rat RNase A is similar to that of the other members of the pancreatic ribonuclease family. The structural variations within a loop bordering the active site can be correlated with the subtle differences in the enzymatic activities of bovine and rat ribonucleases for different substrates. The most significant difference in the backbone conformation was observed in the loop 15-25. This loop incorporates the subtilisin cleavage site which is responsible for RNase A to RNase S conversion in the bovine enzyme. The rat enzyme does not get cleaved under identical conditions. Molecular docking of this region of the rat enzyme in the active site of subtilisin shows steric incompatibility, although the bovine pancreatic ribonuclease A appropriately fits into this active site. It is therefore inferred that the local conformation of the substrate governs the specificity of subtilisin.
Asunto(s)
Páncreas/enzimología , Ribonucleasa Pancreática/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Perros , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleasa Pancreática/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
A novel computer modeling approach suitable for the structure analysis of small bioactive peptides has been developed. This approach involves identification of conformational patterns in protein structure data bank based on the sequence homology with the bioactive peptide. The models built on the basis of this homology and having common conformational patterns are analyzed under the structural constraints derived from the activity data of various synthetic analogs of the peptide. Application of this procedure to the gonadotropin-releasing hormone (GnRH) resulted in a library of possible structures for GnRH, 9 among which shared a common beta-turn. Further analysis of the structures containing the beta-turn motif, in the context of the structure-activity data, led to a model for the active conformation of GnRH. The topology of the putative receptor binding site of the hormone is defined by a contiguous surface formed through an appropriate juxtaposition of the N-terminal pGlu1, the guanidyl group of Arg8, aromatic side chain of Trp3, and the Gly10-NH2 at the C-terminal end.