RESUMEN

BACKGROUND: Thermodynamic and kinetic studies of the Protein L B1 domain (Ppl) suggest a folding pathway in which, during the folding transition, the first beta hairpin is formed while the second beta hairpin and the alpha helix are largely unstructured. The same mutations in the two beta turns have opposite effects on the folding and unfolding rates. Three of the four residues composing the second beta turn in Ppl have consecutive positive phi angles, indicating strain in the second beta turn. RESULTS: We have determined the crystal structures of the beta turn mutants G55A, K54G, and G15A, as well as a core mutant, V49A, in order to investigate how backbone strain affects the overall structure of Ppl. Perturbation of the hydrophobic interactions at the closed interface by the V49A mutation triggered the domain swapping of the C-terminal beta strand that relieved the strain in the second beta turn. Interestingly, the asymmetric unit of V49A contains two monomers and one domain-swapped dimer. The G55A mutation escalated the strain in the second beta turn, and this increased strain shifted the equilibrium toward the domain-swapped dimer. The K54G structure revealed that the increased stability is due to the reduction of strain in the second beta turn, while the G15A structure showed that increased strain alone is insufficient to trigger domain swapping. CONCLUSIONS: Domain swapping in Ppl is determined by the balance of two opposing components of the free energy. One is the strain in the second beta turn that favors the dimer, and the other is the entropic cost of dimer formation that favors the monomer. A single-site mutation can disrupt this balance and trigger domain swapping.

Asunto(s)

Proteínas Bacterianas/química , Peptostreptococcus , Pliegue de Proteína , Proteínas Bacterianas/genética , Cristalografía , Dimerización , Enlace de Hidrógeno , Cinética , Modelos Químicos , Modelos Moleculares , Mutación , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Termodinámica

RESUMEN

Protein L consists of a single alpha-helix packed on a four-stranded beta-sheet formed by two symmetrically opposed beta-hairpins. We use a computer-based protein design procedure to stabilize a domain-swapped dimer of protein L in which the second beta-turn straightens and the C-terminal strand inserts into the beta-sheet of the partner. The designed obligate dimer contains three mutations (A52V, N53P, and G55A) and has a dissociation constant of approximately 700 pM, which is comparable to the dissociation constant of many naturally occurring protein dimers. The structure of the dimer has been determined by x-ray crystallography and is close to the in silico model.

Asunto(s)

Proteínas Bacterianas , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Dimerización , Guanidina , Modelos Moleculares , Mutagénesis , Desnaturalización Proteica , Estructura Secundaria de Proteína

RESUMEN

Apoptosis is an essential physiological process, regulated by the family of Bcl-2-related proteins. However, the molecular mechanism by which Bcl-2 regulates apoptosis still remains elusive. Here we report the functional studies of recombinant human Bcl-2 with the deletion of 22 residues at the C-terminal membrane-anchoring region (rhBcl-2Delta22). Characterization of rhBcl-2Delta22 showed that the recombinant protein is homogeneous and monodisperse in nondenaturing solutions, stable at room temperature in the presence of a metal chelator, and an alpha-helical protein with unfolding of secondary structure at a T(m) of 62.8 degrees C. Optimal membrane pore formation by rhBcl-2Delta22 required negatively charged phospholipids. The existence of a hydrophobic groove in rhBcl-2Delta22 was demonstrated by the fluorescence enhancement of the hydrophobic ANS probe with which a pro-apoptotic Bak BH3 peptide competed. The respiratory inhibitor antimycin A also bound to the hydrophobic groove of rhBcl-2Delta22 with a K(d) of 0.82 microM. The optimal binding conformation of antimycin A was predicted from molecular docking of antimycin A with the hBcl-2 model created by homology modeling. Antimycin A selectively induces apoptosis in cells overexpressing Bcl-2, suggesting that hydrophobic groove-binding compounds may act as selective apoptotic triggers in tumor cells.

Asunto(s)

Antibacterianos/química , Antimicina A/química , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Naftalenosulfonatos de Anilina/metabolismo , Animales , Antibacterianos/metabolismo , Antimicina A/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular , Dicroismo Circular , Simulación por Computador , Colorantes Fluorescentes/metabolismo , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Humanos , Canales Iónicos/química , Canales Iónicos/metabolismo , Ligandos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Eliminación de Secuencia , Espectrometría de Fluorescencia , Termodinámica , Transfección

RESUMEN

Carbonic anhydrases fall into three distinct evolutionary and structural classes: alpha, beta, and gamma. The beta-class carbonic anhydrases (beta-CAs) are widely distributed among higher plants, simple eukaryotes, eubacteria, and archaea. We have determined the crystal structure of ECCA, a beta-CA from Escherichia coli, to a resolution of 2.0 A. In agreement with the structure of the beta-CA from the chloroplast of the red alga Porphyridium purpureum, the active-site zinc in ECCA is tetrahedrally coordinated by the side chains of four conserved residues. These results confirm the observation of a unique pattern of zinc ligation in at least some beta-CAS: The absence of a water molecule in the inner coordination sphere is inconsistent with known mechanisms of CA activity. ECCA activity is highly pH-dependent in the physiological range, and its expression in yeast complements an oxygen-sensitive phenotype displayed by a beta-CA-deletion strain. The structural and biochemical characterizations of ECCA presented here and the comparisons with other beta-CA structures suggest that ECCA can adopt two distinct conformations displaying widely divergent catalytic rates.

Asunto(s)

Anhidrasas Carbónicas/química , Escherichia coli/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Anhidrasas Carbónicas/aislamiento & purificación , Anhidrasas Carbónicas/metabolismo , Cristalografía por Rayos X , Eliminación de Gen , Prueba de Complementación Genética , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Relación Estructura-Actividad , Zinc/metabolismo

RESUMEN

The three-dimensional structure of a tryptophan-containing variant of the IgG-binding B1 domain of protein L has been solved in two crystal forms to 1.7 and 1.8 A resolution. In one of the crystal forms, the entire N-terminal histidine-tag region was immobilized through the coordination of zinc ions and its structural conformation along with the zinc coordination scheme were determined. However, the ordering of the histidine tag by zinc does not affect the overall structure of the rest of the protein. Structural comparisons of the tryptophan-containing variant with an NMR-derived wild-type structure, which contains a tyrosine at position 47, reveals a common fold, although the overall backbone root-mean-square difference is 1.5 A. The Y47W substitution only caused local rearrangement of several side chains, the most prominent of which is the rotation of the Tyr34 side chain, resulting in a 6 A displacement of its hydroxyl group. A small methyl-sized cavity bounded by beta-strands 1, 2 and 4 and the alpha-helix was found in the structures of the Y47W-substituted protein L B1 domain. This cavity may be created as the result of subsequent side-chain rearrangements caused by the Y47W substitution. These high-resolution structures of the tryptophan-containing variant provide a reference frame for the analysis of thermodynamic and kinetic data derived from a series of mutational studies of the protein L B1 domain.

Asunto(s)

Sustitución de Aminoácidos/genética , Proteínas Bacterianas , Proteínas de Unión al ADN/química , Histidina , Peptostreptococcus/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Resonancia Magnética Nuclear Biomolecular , Péptidos/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Termodinámica , Triptófano/genética , Triptófano/metabolismo , Tirosina/genética , Tirosina/metabolismo , Zinc/química , Zinc/metabolismo

RESUMEN

Carbonic anhydrases are zinc metalloenzymes that fall into three distinct evolutionary and structural classes, alpha, beta and gamma. Although alpha-class enzymes, particularly mammalian carbonic anhydrase II, have been the subject of extensive structural studies, for the beta class, consisting of a wide variety of prokaryotic and plant chloroplast carbonic anhydrases, the structural data is quite limited. A member of the beta class from E. coli (CynT2) has been crystallized in native and selenomethionine-labelled forms and multiwavelength anomalous dispersion techniques have been applied in order to determine the positions of anomalous scatterers. The resulting phase information is sufficient to produce an interpretable electron-density map. A crystal structure for CynT2 would contribute significantly to the emerging structural knowledge of a biologically important class of enzymes that perform critical functions in carbon fixation and prokaryotic metabolism.

Asunto(s)

Proteínas Bacterianas/química , Anhidrasas Carbónicas/química , Escherichia coli/enzimología , Isoenzimas/química , Proteínas Bacterianas/genética , Anhidrasas Carbónicas/genética , Cationes , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Isoenzimas/genética , Selenio/química , Selenometionina/química , Zinc/química

RESUMEN

The small 62-residue IgG-binding domain B1 of protein L from Peptostreptococcus magnus (Ppl-B1) has proven to be a simple system for the study of the thermodynamics and kinetics of protein folding. X-ray diffraction studies have been initiated in order to determine how the thermostability, folding and unfolding rates of a series of point mutations spanning Ppl-B1 correlate with the high-resolution structures. To this end, a tryptophan-containing variant of Ppl-B1 (herein known as wild type) and two mutants, Lys61Ala and Val49Ala, have been crystallized. Full data sets have been collected for the wild type and the Lys61Ala and Val49Ala mutants to resolutions of 1. 7, 2.3 and 1.8 A, respectively. Interestingly, all three crystallize using different precipitants and in different space groups. This may be a consequence of the relatively large effects of single-site mutations on surface-charge distribution or structural conformation, which might affect crystal contact sites.

Asunto(s)

Proteínas Bacterianas/química , Alanina , Sustitución de Aminoácidos , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Sitios de Unión de Anticuerpos , Cristalización , Cristalografía por Rayos X , Inmunoglobulina G , Lisina , Mutagénesis Sitio-Dirigida , Peptostreptococcus/química , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Valina

RESUMEN

Patterned expression of the Drosophila rhomboid (rho) gene is thought to promote signaling by the EGF receptor (EGFR) in specific cell types. In this report we examine the subcellular localization of the Rhomboid protein (Rho) which is predicted to be an integral membrane protein. At the light level, immunocytochemical staining for Rho reveals a small number of large patches (or plaques) at or near the apical cell surface. In some cells Rho plaques colocalize with Armadillo at adherens junctions, while in other cells plaques are only found basal to the adherens junction. Immunoelectron microscopy reveals that Rho plaques are composed of a highly localized patch of plasma membrane and a densely staining underlying structure. Concentration of Rho in distinct plaques depends on a balance of synthesis and membrane recycling since increasing the amount of rho expression or blocking membrane recycling leads to more uniform cell surface labeling. A limiting cellular component also appears to be required for concentrating Rho in plaques. We discuss clustering of Rho in plasma membrane patches with respect to the proposed role of Rho in promoting EGF-R signaling.

Asunto(s)

Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Animales , Membrana Celular/metabolismo , Drosophila/ultraestructura , Receptores ErbB/metabolismo , Inmunohistoquímica , Hibridación in Situ , Proteínas de la Membrana/biosíntesis , Microscopía Inmunoelectrónica , Transducción de Señal , Transcripción Genética

RESUMEN

We describe a double-label in situ hybridization protocol based on the optimized synthesis of biotin-labeled RNA probes. Biotin-labeled probes are used in conjunction with digoxigenin-labeled probes to simultaneously visualize two different transcripts. One transcript is hybridized with a biotin-labeled RNA probe and visualized as a brown peroxidase reaction product, and the other transcript is hybridized with a digoxigenin-labeled RNA probe and visualized as a blue alkaline phosphatase reaction product. We present several examples in which this double-labeling method has proven useful in determining the spatial and temporal relationships between various transcripts expressed during Drosophila embryogenesis, indicating that this method should be of general use in establishing the relationship of two independent transcription patterns.

Asunto(s)

Biotina , Digoxigenina , Hibridación in Situ/métodos , Sondas ARN

RESUMEN

Pattern formation in the dorsal region of the Drosophila embryo depends on the activity of a small group of zygotically acting genes. dpp, a key gene in this group, encodes a TGF-beta-like product (Dpp) that has been proposed to function as a morphogen with peak levels of Dpp-specifying amnioserosa, the dorsal-most cell type, and lower Dpp levels specifying dorsal ectoderm. The short gastrulation gene also contributes to patterning the dorsal region, but unlike the other genes involved in this process, sog activity is only required in ventral cells. Genetic evidence indicates that sog functions to antagonize dpp activity. In this report we present further phenotypic characterization of sog mutant embryos in dorsal and lateral regions and describe the cloning of the sog locus. sog is expressed in a broad lateral stripe of cells that abuts the dorsal territory of dpp-expressing cells. sog is predicted to encode a protein with an internal signal sequence and a large extracellular domain containing four repeats of a novel motif defined by the spacing of 10 cysteine residues that is distantly related to domains present in thrombospondin and procollagen. We propose that one or more of these cysteine repeats can be liberated by proteolytic cleavage of the primary Sog protein. These putative soluble Sog peptides may then diffuse into the dorsal region to antagonize the activity of Dpp, leading to the subdivision of the dorsal territory into amnioserosa and dorsal ectoderm.

Asunto(s)

Proteínas de Drosophila , Drosophila/embriología , Drosophila/genética , Gástrula/fisiología , Expresión Génica , Genes de Insecto , Hormonas de Insectos/fisiología , Factor de Crecimiento Transformador beta/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Gástrula/citología , Regulación de la Expresión Génica , Genes Letales , Heterocigoto , Hormonas de Insectos/genética , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Factor de Crecimiento Transformador beta/genética

RESUMEN

Internalization of ligand-receptor complexes is a well-documented mechanism for limiting the duration and magnitude of a signaling event. In the case of the EGF-Receptor (EGF-R), exposure to EGF or TGF-alpha results in internalization of up to 95% of the surface receptor pool within 5 minutes of exposure to ligand. In this report, we show that levels of Drosophila Egf-r mRNA are strongly down-regulated in epidermal cells likely to have recently undergone high levels of EGF-R signaling. The cells in which Egf-r mRNA levels are down-regulated express the rhomboid gene, which is thought to locally amplify EGF-R signaling. Widespread Egf-r mRNA down-regulation can be induced by ubiquitous expression of rhomboid or by eliminating the Gap1 gene. These results suggest that cells engaged in intense EGF-R/RAS signaling limit the duration of the signal through a combination of short-acting negative feedback mechanisms such as receptor internalization followed by a longer lasting reduction in receptor transcript levels. Control of Egf-r mRNA levels by altering transcription or mRNA stability is a new tier of regulation to be considered in analysis of EGF-R signaling during development.

Asunto(s)

Proteínas de Drosophila , Drosophila/embriología , Receptores ErbB/genética , Proteínas Quinasas , Receptores de Péptidos de Invertebrados/genética , Transducción de Señal/fisiología , Animales , Regulación hacia Abajo , Drosophila/genética , Retroalimentación , Hibridación in Situ , Hormonas de Insectos/genética , Proteínas de la Membrana/genética , ARN Mensajero/análisis , Transcripción Genética