RESUMEN

Recent breakthroughs in the structural biology of cytoskeletal motor proteins show that two distinct families of motors--kinesins and myosins - use a similar mechanism of conformational switching for converting small structural changes in their nucleotide-binding sites into larger movements to provide force generation and motion. This mechanism is found to be similar to that employed by G proteins, the well-known molecular switches that regulate protein-protein interactions in many biological systems.

Asunto(s)

Cinesinas/fisiología , Proteínas Motoras Moleculares/fisiología , Miosinas/fisiología , Dominio Catalítico , Predicción , Nucleótidos , Conformación Proteica

RESUMEN

We report on 54 Spanish patients with McArdle's disease from 40 unrelated families. Molecular analysis revealed that the most common R49X mutation was present in 70% of patients and 55% of alleles. The G204S mutation was less frequent and found in 14.8% of patients and 9% of mutant alleles. The W797R mutation was observed in 16.5% of patients, accounting for 13.7% of mutant alleles. Moreover, 78% of mutant alleles among Spanish patients can be identified by using polymerase chain reaction-restriction fragment length polymorphism analysis for the R49X, G204S, and W797R mutations, which makes noninvasive diagnosis possible through molecular genetic analysis of blood DNA. Six novel mutations were found. Three were missense mutations, E348K, R601W, and A703V; two nonsense mutations, E124X and Q754X; and one single base pair deletion, 533 delA. No clear genotype-phenotype correlation emerges from our study. Most of the mutations of uncharged and solvent inaccessible residues and the truncations must disrupt the basic structure of the protein. The mutations of charged residues would be expected to interfere with internal hydrogen bonding networks, introducing severe incompatible partnering that is caused by poor packing or electrostatic repulsions.

Asunto(s)

Glucógeno Fosforilasa de Forma Muscular/deficiencia , Glucógeno Fosforilasa de Forma Muscular/genética , Enfermedad del Almacenamiento de Glucógeno Tipo V/enzimología , Enfermedad del Almacenamiento de Glucógeno Tipo V/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Sitios de Unión/genética , Niño , Femenino , Pruebas Genéticas , Genotipo , Enfermedad del Almacenamiento de Glucógeno Tipo V/epidemiología , Heterocigoto , Homocigoto , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación , Fenotipo , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , España/epidemiología

RESUMEN

The protease inhibitor ecotin fails to inhibit thrombin despite its broad specificity against serine proteases. A point mutation (M84R) in ecotin results in a 1.5 nM affinity for thrombin, 10(4) times stronger than that of wild-type ecotin. The crystal structure of bovine thrombin is determined in complex with ecotin M84R mutant at 2.5 A resolution. Surface loops surrounding the active site cleft of thrombin have undergone significant structural changes to permit inhibitor binding. Particularly, the insertion loops at residues 60 and 148 in thrombin, which likely mediate the interactions with macromolecules, are displaced when the complex forms. Thrombin and ecotin M84R interact in two distinct surfaces. The loop at residue 99 and the C-terminus of thrombin contact ecotin through mixed polar and nonpolar interactions. The active site of thrombin is filled with eight consecutive amino acids of ecotin and demonstrates thrombin's preference for specific features that are compatible with the thrombin cleavage site: negatively charged-Pro-Val-X-Pro-Arg-hydrophobic-positively charged (P1 Arg is in bold letters). The preference for a Val at P4 is clearly defined. The insertion at residue 60 may further affect substrate binding by moving its adjacent loops that are part of the substrate recognition sites.

Asunto(s)

Proteínas Bacterianas/química , Proteínas de Escherichia coli , Proteínas Periplasmáticas , Inhibidores de Serina Proteinasa/química , Trombina/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Bovinos , Cristalografía por Rayos X , Modelos Moleculares , Mutación Puntual , Conformación Proteica , Estructura Secundaria de Proteína , Sensibilidad y Especificidad , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Trombina/metabolismo

RESUMEN

Using recombinant fibronectin proteins containing the V region and two point mutations in the high-affinity heparin-binding domain, we previously showed that these domains modulate tumor cell invasion as well as proteinase expression and apoptosis in human fibroblasts. Structurally, the wildtype counterparts to these two point mutations, together with four other discontinuous, positively charged residues, form a cationic cradle in domain III-13 of fibronectin that binds heparin. We constructed a three-dimensional model of this cationic cradle and determined whether the two engineered point mutations in the heparin-binding domain would alter this cradle conformation, thus explaining the altered cell behavior. Our model of fibronectin domain III-13 was generated from a template of the three-dimensional structure of a homologous (25% identity) domain, III-3, from tenascin. The amino acid sequences of III-13 that differed from tenascin III-3 were replaced, and side chains for positively charged arginines 6 and 7 were substituted with uncharged threonines. The model revealed that the two mutated threonine residues were solvent accessible, readily accommodated as part of an antiparallel beta strand, and remained part of the three-dimensional cradle. These models suggest that the two point mutations in the heparin-binding domain of fibronectin III-13 alter cell function probably through changes in charge and not through changes in the conformational structure of the cationic cradle. J. Cell. Biochem. Suppl. 36: 156-161, 2001.

Asunto(s)

Fibronectinas/química , Heparina/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Fibronectinas/genética , Fibronectinas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química

RESUMEN

Thyroid hormones have some actions that might be useful therapeutically, but others that are deleterious. Potential therapeutically useful actions include those to induce weight loss and lower plasma cholesterol levels. Potential deleterious actions are those on the heart to induce tachycardia and arrhythmia, on bone to decrease mineral density, and on muscle to induce wasting. There have been successes in selectively modulating the actions of other classes of hormones through various means, including the use of pharmaceuticals that have enhanced affinities for certain receptor isoforms. Thus, there is reason to pursue selective modulation of thyroid hormone receptor (TR) function, and several agents have been shown to have some beta-selective, hepatic selective and/or cardiac sparring activities, although development of these was largely not based on detailed understanding of mechanisms for the specificity. The possibility of selectively targeting the TRbeta was suggested by the findings that there are alpha- and beta-TR forms and that the TRalpha-forms may preferentially regulate the heart rate, whereas many other actions of these hormones are mediated by the TRbeta. We determined X-ray crystal structures of the TRalpha and TRbeta ligand-binding domains (LBDs) complexed with the thyroid hormone analog 3,5,3'-triiodithyroacetic acid (Triac). The data suggested that a single amino acid difference in the ligand-binding cavities of the two receptors could affect hydrogen bonding in the receptor region, where the ligand's 1-position substituent fits and might be exploited to generate beta-selective ligands. The compound GC-1, with oxoacetate in the 1-position instead of acetate as in Triac, exhibited TRbeta-selective binding and actions in cultured cells. An X-ray crystal structure of the GC-1-TRbeta LBD complex suggests that the oxoacetate does participate in a network of hydrogen bonding in the TR LBD polar pocket. GC-1 displayed actions in tadpoles that were TRbeta-selective. When administered to mice, GC-1 was as effective in lowering plasma cholesterol levels as T(3), and was more effective than T(3) in lowering plasma triglyceride levels. At these doses, GC-1 did not increase the heart rate. GC-1 was also less active than T(3) in modulating activities of several other cardiac parameters, and especially a cardiac pacemaker channel such as HCN-2, which may participate in regulation of the heart rate. GC-1 showed intermediate activity in suppressing plasma thyroid stimulating hormone (TSH) levels. The tissue/plasma ratio for GC-1 in heart was also less than for the liver. These data suggest that compounds can be generated that are TR-selective and that compounds with this property and/or that exhibit selective uptake, might have clinical utility as selective TR modulators.

Asunto(s)

Receptores de Hormona Tiroidea/fisiología , Animales , Humanos , Isoformas de Proteínas/efectos de los fármacos , Isoformas de Proteínas/fisiología , Receptores de Hormona Tiroidea/efectos de los fármacos

RESUMEN

Kinesin motors are specialized enzymes that use hydrolysis of ATP to generate force and movement along their cellular tracks, the microtubules. Although numerous biochemical and biophysical studies have accumulated much data that link microtubule-assisted ATP hydrolysis to kinesin motion, the structural view of kinesin movement remains unclear. This study of the monomeric kinesin motor KIF1A combines X-ray crystallography and cryo-electron microscopy, and allows analysis of force-generating conformational changes at atomic resolution. The motor is revealed in its two functionally critical states-complexed with ADP and with a non-hydrolysable analogue of ATP. The conformational change observed between the ADP-bound and the ATP-like structures of the KIF1A catalytic core is modular, extends to all kinesins and is similar to the conformational change used by myosin motors and G proteins. Docking of the ADP-bound and ATP-like crystallographic models of KIF1A into the corresponding cryo-electron microscopy maps suggests a rationale for the plus-end directional bias associated with the kinesin catalytic core.

Asunto(s)

Cinesinas/fisiología , Proteínas Motoras Moleculares , Proteínas del Tejido Nervioso/fisiología , Adenosina Difosfato/fisiología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/química , Adenosina Trifosfato/fisiología , Dominio Catalítico , Microscopía por Crioelectrón , Cristalografía por Rayos X , Cinesinas/química , Microtúbulos/fisiología , Modelos Biológicos , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Conformación Proteica , Relación Estructura-Actividad

RESUMEN

Species and tissue-specific isozymes of phosphorylase display differences in regulatory properties consistent with their distinct roles in particular organisms and tissues. In this review, we compare crystallographic structures of regulated and unregulated phosphorylases, including maltodextrin phosphorylase (MalP) from Escherichia coli, glycogen phosphorylase from yeast, and mammalian isozymes from muscle and liver tissues. Mutagenesis and functional studies supplement the structural work and provide insights into the structural basis for allosteric control mechanisms. MalP, a simple, unregulated enzyme, is contrasted with the more complicated yeast and mammalian phosphorylases that have evolved regulatory sites onto the basic catalytic architecture. The human liver and muscle isozymes show differences structurally in their means of invoking allosteric activation. Phosphorylation, though common to both the yeast and mammalian enzymes, occurs at different sites and activates the enzymes by surprisingly different mechanisms.

Asunto(s)

Fosforilasas/química , Fosforilasas/metabolismo , Sitio Alostérico , Animales , Dimerización , Glucosiltransferasas/química , Humanos , Isoenzimas , Hígado/enzimología , Modelos Moleculares , Músculos/enzimología , Mutagénesis Sitio-Dirigida , Fosforilasas/genética , Fosforilación , Pliegue de Proteína , Estructura Terciaria de Proteína , Difracción de Rayos X

RESUMEN

Success of mitosis depends upon the coordinated and regulated activity of many cellular factors, including kinesin motor proteins, which are required for the assembly and function of the mitotic spindle. Eg5 is a kinesin implicated in the formation of the bipolar spindle and its movement prior to and during anaphase. We have determined the crystal structure of the Eg5 motor domain with ADP-Mg bound. This structure revealed a new intramolecular binding site of the neck-linker. In other kinesins, the neck-linker has been shown to be a critical mechanical element for force generation. The neck-linker of conventional kinesin is believed to undergo an ordered-to-disordered transition as it translocates along a microtubule. The structure of Eg5 showed an ordered neck-linker conformation in a position never observed previously. The docking of the neck-linker relies upon residues conserved only in the Eg5 subfamily of kinesin motors. Based on this new information, we suggest that the neck-linker of Eg5 may undergo an ordered-to-ordered transition during force production. This ratchet-like mechanism is consistent with the biological activity of Eg5.

Asunto(s)

Cinesinas/química , Proteínas de Xenopus , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Clonación Molecular , Secuencia Conservada , Cristalografía por Rayos X , Humanos , Cinesinas/genética , Mitosis , Modelos Moleculares , Datos de Secuencia Molecular , Placenta/química , Conformación Proteica

RESUMEN

Ecotin is a homodimeric protein from Escherichia coli that inhibits many serine proteases of the chymotrypsin fold, often with little effect from the character or extent of enzyme substrate specificity. This pan-specificity of inhibition is believed to derive from formation of a heterotetrameric complex with target proteases involving three types of interface: the dimerization interface, a primary substrate-like interaction, and a smaller secondary interaction between the partner ecotin subunit and the protease. A monomeric ecotin variant (mEcotin) and a single-chain ecotin dimer (scEcotin) were constructed to study the effect of a network of protein interactions on binding affinity and the role of dimerization in broad inhibitor specificity. mEcotin was produced by inserting a beta-turn into the C-terminal arm, which normally exchanges with the other subunit. While the dimerization constant (K(dim)) of wild-type (WT) ecotin was found to be picomolar by subunit exchange experiments using FRET and by association kinetics, mEcotin was monomeric up to 1 mM as judged by gel filtration and analytical centrifugation. A crystal structure of uncomplexed mEcotin to 2.0 A resolution verifies the design, showing a monomeric protein in which the C-terminal arm folds back onto itself to form a beta-barrel structure nearly identical to its dimeric counterpart. The kinetic rate constants and equilibrium dissociation constants for monomeric and dimeric ecotin variants were determined with both trypsin and chymotrypsin. The effect of the secondary binding site on affinity was found to vary inversely with the strength of the interaction at the primary site. This compensatory effect yields a nonadditivity of up to 5 kcal/mol and can be explained in terms of the optimization of binding orientation. Such a mechanism of adaptability allows femtomolar affinities for two proteases with very different specificities.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas Periplasmáticas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Sitios de Unión , Quimotripsina/metabolismo , Cristalografía por Rayos X , Dimerización , Fluorescencia , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Peso Molecular , Mutación/genética , Unión Proteica , Ingeniería de Proteínas , Estructura Cuaternaria de Proteína , Subunidades de Proteína , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/farmacología , Especificidad por Sustrato , Termodinámica , Tripsina/metabolismo

RESUMEN

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia. We previously determined the crystal structure of the rat (r) TRalpha ligand-binding domain (LBD). In the present study, we determined the crystal structure of the rTRalpha LBD in a complex with an additional ligand, Triac (3,5, 3'-triiodothyroacetic acid), and two crystal structures of the human (h) TRbeta receptor LBD in a complex with either Triac or a TRbeta-selective compound, GC-1 [3,5-dimethyl-4-(4'-hydroy-3'-isopropylbenzyl)-phenoxy acetic acid]. The rTRalpha and hTRbeta LBDs show close structural similarity. However, the hTRbeta structures extend into the DNA-binding domain and allow definition of a structural "hinge" region of only three amino acids. The two TR subtypes differ in the loop between helices 1 and 3, which could affect both ligand recognition and the effects of ligand in binding coactivators and corepressors. The two subtypes also differ in a single amino acid residue in the hormone-binding pocket, Asn (TRbeta) for Ser (TRalpha). Studies here with TRs in which the subtype-specific residue is exchanged suggest that most of the selectivity in binding derives from this amino acid difference. The flexibility of the polar region in the TRbeta receptor, combined with differential recognition of the chemical group at the 1-carbon position, seems to stabilize the complex with GC-1 and contribute to its beta-selectivity. These results suggest a strategy for development of subtype-specific compounds involving modifications of the ligand at the 1-position.

Asunto(s)

Receptores de Hormona Tiroidea/química , Receptores de Hormona Tiroidea/metabolismo , Triyodotironina/análogos & derivados , Acetatos/química , Acetatos/metabolismo , Secuencia de Aminoácidos , Asparagina , Sitios de Unión , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Mutación , Fenoles/química , Fenoles/metabolismo , Conformación Proteica , Receptores de Hormona Tiroidea/genética , Homología de Secuencia de Aminoácido , Hormonas Tiroideas/metabolismo , Triyodotironina/química , Triyodotironina/metabolismo

RESUMEN

Thyroid hormone receptors (TRs) bind as homodimers or heterodimers with retinoid X receptors (RXRs) to DNA elements with diverse orientations of AGGTCA half-sites. We performed a comprehensive x-ray crystal structure-guided mutation analysis of the TR ligand binding domain (TR LBD) surface to map the functional interface for TR homodimers and heterodimers with RXR in the absence and/or in the presence of DNA. We also identified the molecular contacts in TR LBDs crystallized as dimers. The results show that crystal dimer contacts differ from those found in the functional studies. We found that identical TR LBD residues found in helices 10 and 11 are involved in TR homodimerization and heterodimerization with RXR. Moreover, the same TR LBD surface is operative for dimerization with direct repeats spaced by 4 base pairs (DR-4) and with the inverted palindrome spaced by 6 base pairs (F2), but not with TREpal (unspaced palindrome), where homodimers appear to be simply two monomers binding independently to DNA. We also demonstrate that interactions between the TR and RXR DNA binding domains stabilize TR-RXR heterodimers on DR-4. The dimer interface can be functional in the cell, because disruption of key residues impairs transcriptional activity of TRs mediated through association with RXR LBD linked to GAL4 DNA-binding domain.

Asunto(s)

Receptores de Ácido Retinoico/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Línea Celular , Cristalización , Dimerización , Ligandos , Modelos Moleculares , Conformación Proteica , Receptores de Hormona Tiroidea/química , Receptores X Retinoide

RESUMEN

BACKGROUND: Cysteine proteases of the papain superfamily are present in nearly all groups of eukaryotes and play vital roles in a wide range of biological processes and diseases, including antigen and hormone processing, bacterial infection, arthritis, osteoporosis, Alzheimer's disease and cancer-cell invasion. Because they are critical to the life-cycle progression of many pathogenic protozoa, they represent potential targets for selective inhibitors. Chagas' disease, the leading cause of death due to heart disease in Latin American countries, is transmitted by Trypanosoma cruzi. Cruzain is the major cysteine protease of T cruzi and has been the target of extensive structure-based drug design. RESULTS: High-resolution crystal structures of cruzain bound to a series of potent phenyl-containing vinyl-sulfone, sulfonate and sulfonamide inhibitors have been determined. The structures show a consistent mode of interaction for this family of inhibitors based on a covalent Michael addition formed at the enzyme's active-site cysteine, hydrophobic interactions in the S2 substrate-binding pocket and a strong constellation of hydrogen bonding in the S1' region. CONCLUSIONS: The series of vinyl-sulfone-based inhibitors examined in complex with cruzain was designed to probe recognition and binding potential of an aromatic-rich region of the enzyme. Analysis of the interactions formed shows that aromatic interactions play a less significant role, whereas the strength and importance of hydrogen bonding in the conformation adopted by the inhibitor upon binding to the enzyme was highlighted. A derivative of one inhibitor examined is currently under development as a therapeutic agent against Chagas' disease.

Asunto(s)

Enfermedad de Chagas/enzimología , Cisteína Endopeptidasas/química , Conformación Proteica , Proteínas Protozoarias/química , Trypanosoma/química , Animales , Sitios de Unión , Cisteína Endopeptidasas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Protozoarias/metabolismo , Relación Estructura-Actividad , Especificidad por Sustrato , Trypanosoma/enzimología

RESUMEN

Granzyme B is a serine protease of the chymotrypsin fold that mediates cell death by cytotoxic lymphocytes. It is a processing enzyme, requiring extended peptide substrates containing an Asp residue. The determinants that allow for this substrate specificity are revealed in the three-dimensional structure of granzyme B in complex with a macromolecular inhibitor. The primary specificity for Asp occurs through a side-on interaction with Arg 226, a buried Arg side chain of granzyme B. An additional nine amino acids make contact with the substrate and define the granzyme B extended substrate specificity profile. The substrate determinants found in this structure are shared by other members of this protein class and help to reveal the properties that define substrate specificity.

Asunto(s)

Apoptosis , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Proteínas Periplasmáticas , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Ácido Aspártico/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Cristalografía por Rayos X , Glicosilación , Granzimas , Humanos , Modelos Moleculares , Mutación , Conformación Proteica , Ratas , Alineación de Secuencia , Serina Endopeptidasas/genética , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Especificidad por Sustrato

RESUMEN

Kinesin, a microtubule-based motor, and myosin, an actin-based motor, share a similar core structure, indicating that they arose from a common ancestor. However, kinesin lacks the long lever-arm domain that is believed to drive the myosin power stroke. Here, we present evidence that a much smaller region of ca. 10-40 amino acids serves as a mechanical element for kinesin motor proteins. These 'neck regions' are class conserved and have distinct structures in plus-end and minus-end-directed kinesin motors. Mutagenesis studies also indicate that the neck regions are involved in coupling ATP hydrolysis and energy into directional motion along the microtubule. We suggest that the kinesin necks drive motion by undergoing a conformational change in which they detach and re-dock onto the catalytic core during the ATPase cycle. Thus, kinesin and myosin have evolved unique mechanical elements that amplify small, nucleotide-dependent conformational changes that occur in their similar catalytic cores.

Asunto(s)

Cinesinas/química , Cinesinas/fisiología , Proteínas Motoras Moleculares/fisiología , Movimiento/fisiología , Animales , Modelos Moleculares , Proteínas Motoras Moleculares/química , Contracción Muscular , Miosinas/química , Miosinas/fisiología , Conformación Proteica

RESUMEN

Ecotin is a dimeric serine protease inhibitor from Escherichia coli which binds proteases to form a hetero-tetramer with three distinct interfaces: an ecotin-ecotin dimer interface, a larger primary ecotin-protease interface, and a smaller secondary ecotin-protease interface. The contributions of these interfaces to binding and inhibition are unequal. To investigate the contribution and adaptability of each interface, we have solved the structure of two mutant ecotin-trypsin complexes and compared them to the structure of the previously determined wild-type ecotin-trypsin complex. Wild-type ecotin has an affinity of 1 nM for trypsin, while the optimized mutant, ecotin Y69F, D70P, which was found using phage display technologies, inhibits rat trypsin with a K(i) value of 0.08 nM. Ecotin 67-70A, M84R which has four alanine substitutions in the ecotin-trypsin secondary binding site, along with the M84R mutation at the primary site, has a K(i) value against rat trypsin of 0.2 nM. The structure of the ecotin Y69F, D70P-trypsin complex shows minor structural changes in the ecotin-trypsin tetramer. The structure of the ecotin 67-70A, M84R mutant bound to trypsin shows large deviations in the tertiary and quaternary structure of the complex. The trypsin structure shows no significant changes, but the conformation of several loop regions of ecotin are altered, resulting in the secondary site releasing its hold on trypsin. The structure of several regions previously considered to be rigid is also significantly modified. The inherent flexibility of ecotin allows it to accommodate these mutations and still maintain tight binding through the compromises of the protein-protein interfaces in the ecotin-trypsin tetramer. A comparison with two recently described ecotin-like genes from other bacteria suggests that these structural and functional features are conserved in otherwise distant bacterial lineages.

Asunto(s)

Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas Periplasmáticas , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/metabolismo , Tripsina/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Animales , Proteínas Bacterianas/genética , Sitios de Unión , Secuencia Conservada , Cristalografía por Rayos X , Dimerización , Evolución Molecular , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Docilidad , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Ratas , Alineación de Secuencia , Inhibidores de Serina Proteinasa/genética , Termodinámica , Tripsina/química , Inhibidores de Tripsina/química , Inhibidores de Tripsina/genética , Inhibidores de Tripsina/metabolismo

RESUMEN

Previous mutagenesis studies along with molecular modeling using the x-ray coordinates of the rabbit 15-lipoxygenase have led to the suggestion that the size of the substrate binding pocket may play an essential role in determining the oxygenation specificity of 5-, 12-, and 15-lipoxygenases. Based on the x-ray crystal structure of rabbit 15-lipoxygenase, Ile(593) appeared to be important in defining size and shape of the substrate-binding site in 15-lipoxygenases. We found that substitution of Ile(593) with alanine shifted the positional specificity of this enzyme toward 12-lipoxygenation. To compare the importance of position 593 with previously defined determinants for the oxygenation specificity, we introduced small (alanine-scan) or large amino acids (phenylalanine-scan) at critical positions surrounding the putative fatty acid-binding site, so that the volume of the pocket was either increased or decreased. Enlargement or alteration in packing density within the substrate binding pocket in the rabbit 15-lipoxygenase increased the share of 12-lipoxygenase products, whereas a smaller active site favored 15-lipoxygenation. Simultaneous substitution of both large and small residues in the context of either a 15- or 12-lipoxygenase indicated that there is a functional interplay of the sequence determinants for lipoxygenation specificity. If the 15-lipoxygenase active site is enlarged excessively, however, no lipoxygenation was observed anymore. Together these results indicate the importance of the overall size and shape of the arachidonic acid binding pocket in defining the specificity of lipoxygenase reaction.

Asunto(s)

Araquidonato 15-Lipooxigenasa/química , Animales , Sitios de Unión , Mutagénesis Sitio-Dirigida , Conejos , Relación Estructura-Actividad , Especificidad por Sustrato

RESUMEN

Ncd is a microtubule minus-end directed motor of the kinesin superfamily. Previously it has been shown that ncd and kinesin motor domains share the same major binding site on microtubules. Here we report a three-dimensional EM reconstruction of negatively stained two-dimensional Zn-induced tubulin crystal sheets (Zn-sheets) decorated with the ncd motor domain at a resolution of 16 A. This work has revealed a second specific binding site for the ncd motor domain. The motor binding site on the tubulin Zn-sheets spans both alpha and beta tubulin subunits. This binding site is located at a position different from the previously identified ncd binding site on microtubules and may play a role in motor function.

Asunto(s)

Proteínas de Drosophila , Cinesinas/química , Tubulina (Proteína)/química , Animales , Sitios de Unión , Cristalografía/métodos , Drosophila , Electrones , Microscopía Electrónica , Microtúbulos/química , Modelos Moleculares , Conformación Proteica , Zinc/química

RESUMEN

The X-ray crystallographic structures of the anti-Syrian hamster prion protein (SHaPrP) monoclonal Fab 3F4 alone, as well as the complex with its cognate peptide epitope (SHaPrP 104-113), have been determined to atomic resolution. The conformation of the decapeptide is an Omega-loop. There are substantial alterations in the antibody combining region upon epitope binding. The peptide binds in a U-shaped groove on the Fab surface, with the two specificity determinants, Met109 and Met112, penetrating deeply into separate hydrophobic cavities formed by the heavy and light chain complementarity-determining regions. In addition to the numerous contacts between the Fab and the peptide, two intrapeptide hydrogen bonds are observed, perhaps indicating the structure bound to the Fab exists transiently in solution. This provides the first structural information on a portion of the PrP N-terminal region observed to be flexible in the NMR studies of SHPrP 90-231, SHaPrP 29-231 and mouse PrP 23-231. Antibody characterization of the antigenic surfaces of PrPC and PrPSc identifies this flexible region as a component of the conformational rearrangement that is an essential feature of prion disease.

Asunto(s)

Anticuerpos Monoclonales/inmunología , Epítopos/química , Proteínas PrPC/química , Proteínas PrPC/inmunología , Proteínas PrPSc/química , Proteínas PrPSc/inmunología , Animales , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos/inmunología , Sitios de Unión , Cricetinae , Cristalización , Cristalografía por Rayos X , Epítopos/inmunología , Epítopos/metabolismo , Enlace de Hidrógeno , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Mesocricetus , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Proteínas PrPC/metabolismo , Proteínas PrPSc/metabolismo , Conformación Proteica

RESUMEN

A three-dimensional model for residues 142-427 of the ligand binding domain (LBD) of the human nuclear receptor for 1alpha, 25-dihydroxy-vitamin D(3) [VDR] has been generated based on the X-ray crystallographic atomic coordinates of the LBD of the rat alpha1 thyroid receptor (TR). The VDR LBD model is an elongated globular shape comprised of an antiparallel alpha-helical triple sandwich topology, made up of 12 alpha-helical elements linked by short loop structures; collectively these structural features are similar to the characteristic secondary and tertiary structures for six nuclear receptors with known X-ray structures. The model has been used to describe the interaction of the conformationally flexible natural hormone, 1alpha,25-dihydroxy-vitamin D(3) [1alpha, 25(OH)(2)D(3)], and a number of related analogs with the VDR LBD. The optimal orientation of the 1alpha,25(OH)(2)D(3) in the LBD is with its A-ring directed towards the interior and its flexible side chain pointing towards and interacting with helix-12, site of the activation function-2 domain (AF-2) of the VDR. Mapping of four natural and one experimental point mutations of the VDR LBD, which result in ligand-related receptor dysfunction, indicates the close proximity of these amino acids to the bound ligand.

Asunto(s)

Modelos Moleculares , Vitamina D/análogos & derivados , Secuencia de Aminoácidos , Animales , Sitios de Unión , Núcleo Celular/química , Cristalografía por Rayos X , Humanos , Modelos Químicos , Datos de Secuencia Molecular , Mutagénesis , Conformación Proteica , Ratas , Receptores de Calcitriol/química , Homología de Secuencia de Aminoácido , Vitamina D/química

RESUMEN

Clathrin is a triskelion-shaped cytoplasmic protein that polymerizes into a polyhedral lattice on intracellular membranes to form protein-coated membrane vesicles. Lattice formation induces the sorting of membrane proteins during endocytosis and organelle biogenesis by interacting with membrane-associated adaptor molecules. The clathrin triskelion is a trimer of heavy-chain subunits (1,675 residues), each binding a single light-chain subunit, in the hub domain (residues 1,074-1,675). Light chains negatively modulate polymerization so that intracellular clathrin assembly is adaptor-dependent. Here we report the atomic structure, to 2.6 A resolution, of hub residues 1,210-1,516 involved in mediating spontaneous clathrin heavy-chain polymerization and light-chain association. The hub fragment folds into an elongated coil of alpha-helices, and alignment analyses reveal a 145-residue motif that is repeated seven times along the filamentous leg and appears in other proteins involved in vacuolar protein sorting. The resulting model provides a three-dimensional framework for understanding clathrin heavy-chain self-assembly, light-chain binding and trimerization.

Asunto(s)

Clatrina/química , Secuencias Repetitivas de Aminoácido , Secuencia de Aminoácidos , Animales , Cristalografía , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica