RESUMEN
Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34â kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-LOX resulted in increased solubility of 12R-LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16â kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5â M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2(1), with unit-cell parameters a = 138.97, b = 266.11, c = 152.26â Å, ß = 101.07°. Based on the calculated Matthews coefficient (3.1â Å(3)â Da(-1)), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4â Å resolution using synchrotron radiation.
Asunto(s)
Araquidonato 12-Lipooxigenasa/química , Chaperonina 10/química , Chaperonina 60/química , Araquidonato 12-Lipooxigenasa/genética , Araquidonato 12-Lipooxigenasa/aislamiento & purificación , Araquidonato 12-Lipooxigenasa/metabolismo , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Expresión Génica , Humanos , Unión ProteicaRESUMEN
Human S100A7 (psoriasin) is overexpressed in inflammatory diseases. The recently discovered, co-evolved hS100A15 is almost identical in sequence and up-regulated with hS100A7 during cutaneous inflammation. The functional role of these closely related proteins for inflammation remains undefined. By generating specific Abs, we demonstrate that hS100A7 and hS100A15 proteins are differentially expressed by specific cell types in the skin. Although highly homologous, both proteins are chemoattractants with distinct chemotactic activity for leukocyte subsets. We define RAGE (receptor for advanced glycation end products) as the hS100A7 receptor, whereas hS100A15 functions through a Gi protein-coupled receptor. hS100A7-RAGE binding, signaling, and chemotaxis are zinc-dependent in vitro, reflecting the previously reported zinc-mediated changes in the hS100A7 dimer structure. When combined, hS100A7 and hS100A15 potentiate inflammation in vivo. Thus, proinflammatory synergism in disease may be driven by the diverse biology of these almost identical proteins that have just recently evolved. The identified S100A7 interaction with RAGE may provide a novel therapeutic target for inflammation.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Quimiotaxis de Leucocito , Inflamación/inmunología , Queratinocitos/inmunología , Proteínas S100/metabolismo , Animales , Proteínas de Unión al Calcio/inmunología , Línea Celular , Humanos , Inflamación/metabolismo , Queratinocitos/citología , Queratinocitos/metabolismo , Subgrupos Linfocitarios , Ratones , Ratones Noqueados , Receptor para Productos Finales de Glicación Avanzada , Receptores Acoplados a Proteínas G/inmunología , Receptores Acoplados a Proteínas G/metabolismo , Receptores Inmunológicos/inmunología , Receptores Inmunológicos/metabolismo , Proteína A7 de Unión a Calcio de la Familia S100 , Proteínas S100/inmunologíaRESUMEN
Transglutaminases (TGs) are Ca(2+)-dependent enzymes capable of catalyzing transamidation of glutamine residues to form intermolecular isopeptide bonds. These enzymes are involved in various biological phenomena, including blood coagulation, wound healing, cell death, tissue repair, and terminal differentiation of keratinocytes. Among the TG-family members, TG5 is one of the latest identified enzymes and therefore the less characterized at the functional level. In this work, we reported that TG5 is proteolytically processed in the baculovirus expression system and in mammal epithelial cells. Similar to other members of the TG family-TG1, TG3, and factor XIIIa -, TG5 full-length enzyme has very low enzymatic activity, while the 53-kDa proteolytically processed form is highly active.
Asunto(s)
Células Epiteliales/enzimología , Transglutaminasas/fisiología , Animales , Baculoviridae/metabolismo , Calcio/química , Línea Celular , Células Epiteliales/metabolismo , Factor XIIIa/química , Glutamina/química , Humanos , Insectos , Queratinocitos/citología , Ratones , Péptidos/química , Transglutaminasas/químicaRESUMEN
The title compound, C(8)H(14)N(2) (2+)·C(12)H(8)N(2)O(8)P(-)·ClO(4) (-), was formed by the reaction of α,α-bis-m-xylenediamine and sodium bis-p-nitro-phenyl-phosphate in the presence of Zn(ClO(4))·6H(2)O in methanol solution. The two amine groups of the m-xylenediammonium ion are each protonated and each hydrogen-bonded to two O atoms of the phosphate anion, which acts as a 1,3-bridge. The ammonium groups are arranged matched face to face and each pair is doubly bridged by two perchlorate ions through hydrogen bonding. In addition, there are also weak C-Hâ¯O inter-actions. Both the N-Hâ¯O and C-Hâ¯O inter-actions are contained in a channel down the a axis. The perchlorate oxygen atoms are disordered over two positions with site occupancy factors of ca 0.7 and 0.3.
Asunto(s)
ADN Recombinante/genética , Transglutaminasas/genética , Animales , ADN Complementario/genética , Epidermis/enzimología , Exones/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , ARN Mensajero/genética , Transglutaminasas/química , Transglutaminasas/metabolismoRESUMEN
The stratum corneum, the outer layer of the epidermis, serves as a protective barrier to isolate the skin from the external environment. Keratinocyte transglutaminase 1 (TGase 1) catalyzes amide crosslinking between glutamine and lysine residues on precursor proteins forming the impermeable layers of the epidermal cell envelopes (CE), the highly insoluble membranous structures of the stratum corneum. Patients with the autosomal recessive skin disorder lamellar ichthyosis (LI) appear to have deficient cross-linking of the cell envelope due to mutations identified in TGase 1, linking this enzyme to LI. In the absence of a crystal structure, molecular modeling was used to generate the structure of TGase 1. We have mapped the known mutations of TGase 1 from our survey obtained from a search of PubMed and successfully predicted the impact of these mutations on LI. Furthermore, we have identified Ca(2+) binding sites and propose that Ca(2+) induces a cis to trans isomerization in residues near the active site as part of the enzyme transamidation activation. Docking experiments suggest that substrate binding subsequently induces the reverse cis to trans isomerization, which may be a significant part of the catalytic process. These results give an interpretation at the molecular level of previously reported mutations and lead to further insights into the structural model of TGase 1, providing a new basis for understanding LI.
Asunto(s)
Ictiosis Lamelar/genética , Ictiosis Lamelar/metabolismo , Transglutaminasas/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/química , Catálisis , Humanos , Queratinocitos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Arf (ADP-ribosylation factor) GAPs (GTPase-activating proteins) are enzymes that catalyse the hydrolysis of GTP bound to the small GTP-binding protein Arf. They have also been proposed to function as Arf effectors and oncogenes. We have set out to characterize the kinetics of the GAP-induced GTP hydrolysis using a truncated form of ASAP1 [Arf GAP with SH3 (Src homology 3) domain, ankyrin repeats and PH (pleckstrin homology) domains 1] as a model. We found that ASAP1 used Arf1-GTP as a substrate with a k(cat) of 57+/-5 s(-1) and a K(m) of 2.2+/-0.5 microM determined by steady-state kinetics and a kcat of 56+/-7 s(-1) determined by single-turnover kinetics. Tetrafluoroaluminate (AlF4-), which stabilizes complexes of other Ras family members with their cognate GAPs, also stabilized a complex of Arf1-GDP with ASAP1. As anticipated, mutation of Arg-497 to a lysine residue affected kcat to a much greater extent than K(m). Changing Trp-479, Iso-490, Arg-505, Leu-511 or Asp-512 was predicted, based on previous studies, to affect affinity for Arf1-GTP. Instead, these mutations primarily affected the k(cat). Mutants that lacked activity in vitro similarly lacked activity in an in vivo assay of ASAP1 function, the inhibition of dorsal ruffle formation. Our results support the conclusion that the Arf GAP ASAP1 functions in binary complex with Arf1-GTP to induce a transition state towards GTP hydrolysis. The results have led us to speculate that Arf1-GTP-ASAP1 undergoes a significant conformational change when transitioning from the ground to catalytically active state. The ramifications for the putative effector function of ASAP1 are discussed.
Asunto(s)
Factor 1 de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Guanosina Trifosfato/metabolismo , Factor 1 de Ribosilacion-ADP/química , Factor 1 de Ribosilacion-ADP/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Catálisis , Humanos , Hidrólisis , Cinética , Metabolismo de los Lípidos , Ratones , Modelos Moleculares , Mutación/genética , Células 3T3 NIH , Unión Proteica , Estructura Terciaria de Proteína , Homología Estructural de ProteínaRESUMEN
ARAP2 is a protein that contains both ArfGAP and RhoGAP domains. We found that it is a phosphatidylinositol (3,4,5)-trisphosphate-dependent Arf6 GAP that binds RhoA-GTP but lacks RhoGAP activity. In agreement with the hypothesis that ARAP2 mediates effects of RhoA, endogenous ARAP2 associated with focal adhesions (FAs) and reduction of ARAP2 expression, by RNAi, resulted in fewer FAs and actin stress fibers (SFs). In cells with reduced levels of endogenous ARAP2, FAs and SFs could be restored with wild-type recombinant ARAP2 but not mutants lacking ArfGAP or Rho-binding activity. Constitutively active Arf6 also caused a loss of SFs. The Rho effector ROKalpha was ineffective in restoring FAs. Conversely, overexpression of ARAP2 did not restore SFs in cells treated with a ROK inhibitor but induced punctate accumulations of paxillin. We conclude that ARAP2 is an Arf6GAP that functions downstream of RhoA to regulate focal adhesion dynamics.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Actinas/metabolismo , Proteínas Portadoras/fisiología , Citoesqueleto/metabolismo , Proteínas Activadoras de GTPasa/fisiología , Proteína de Unión al GTP rhoA/metabolismo , Factor 1 de Ribosilacion-ADP/metabolismo , Factor 6 de Ribosilación del ADP , Proteínas Portadoras/genética , Línea Celular , Adhesiones Focales , Proteínas Activadoras de GTPasa/genética , Humanos , Modelos Moleculares , Mutación , Fosfatos de Fosfatidilinositol/fisiología , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Fibras de Estrés/fisiologíaRESUMEN
Bathing suit ichthyosis (BSI) is a striking and unique clinical form of autosomal recessive congenital ichthyosis characterized by pronounced scaling on the bathing suit areas but sparing of the extremities and the central face. Here we report on a series of 10 BSI patients. Our genetic, ultrastructural and biochemical investigations show that BSI is caused by transglutaminase-1 (TGase-1) deficiency. Altogether, we identified 13 mutations in TGM1-among them seven novel missense mutations and one novel nonsense mutation. Structural modeling for the Tyr276Asn mutation reveals that the residue is buried in the hydrophobic interior of the enzyme and that the hydroxyl side chain of Tyr276 is exposed to solvent in a cavity of the enzyme. Cryosections of healthy skin areas demonstrated an almost normal TGase activity, in contrast to the affected BSI skin, which only showed a cytoplasmic and clearly reduced TGase-1 activity. The distribution of TGase-1 substrates in the epidermis of affected skin corresponded to the situation in TGase-1 deficiency. Interestingly, the expression of TGase-3 and cathepsin D was reduced. Digital thermography validated a striking correlation between warmer body areas and presence of scaling in patients suggesting a decisive influence of the skin temperature. In situ TGase testing in skin of BSI patients demonstrated a marked decrease of enzyme activity when the temperature was increased from 25 to 37 degrees C. We conclude that BSI is caused by TGase-1 deficiency and suggest that it is a temperature-sensitive phenotype.
Asunto(s)
Ictiosis/genética , Transglutaminasas/deficiencia , Transglutaminasas/genética , Adolescente , Adulto , Niño , Preescolar , Mapeo Cromosómico , Codón sin Sentido , Femenino , Humanos , Ictiosis/enzimología , Ictiosis/patología , Lactante , Masculino , Modelos Moleculares , Mutación Missense , Fenotipo , Análisis de Secuencia de ADN , Piel/enzimología , Piel/patología , Temperatura , Transglutaminasas/química , Transglutaminasas/metabolismoRESUMEN
The study of bacteriophage T4 assembly has revealed regulatory mechanisms pertinent not only to viruses but also to macromolecular complexes. The capsid of bacteriophage T4 is composed of the major capsid protein gp23, and a minor capsid protein gp24, which is arranged as pentamers at the vertices of the capsid. In this study the T4 capsid protein gp24 and its mutant forms were overexpressed and purified to homogeneity. The overexpression from plasmid vectors of all the constructs in Escherichia coli yields biologically active protein in vivo as determined by assembly of active virus following infection with inactivated gene 24 mutant viruses. The gp24 mutant was subjected to surface entropy reduction by mutagenesis and reductive alkylation in order to improve its crystallization properties and diffraction quality. To determine if surface mutagenesis targeting would result in diffractable crystals, two glutamate to alanine mutations (E89A,E90A) were introduced. We report here the biochemical observations and consequent mutagenesis experiment that resulted in improvements in the stability, crystallizability and crystal quality of gp24 without affecting the overall folding. Rational modification of the protein surface to achieve crystallization appears promising for improving crystallization behavior and crystal diffracting qualities. The crystal of gp24(E89A,E90A) diffracted to 2.6A resolution compared to wild-type gp24 at 3.80A resolution under the same experimental conditions. Surface mutation proved to be a better method than reductive methylation for improving diffraction quality of the gp24 crystals.
Asunto(s)
Sustitución de Aminoácidos , Proteínas de la Cápside/química , Proteínas de la Cápside/aislamiento & purificación , Escherichia coli , Mutación Missense , Proteínas de la Cápside/biosíntesis , Proteínas de la Cápside/genética , Cristalografía por Rayos X/métodos , Escherichia coli/genética , Estructura Terciaria de Proteína , Ensamble de Virus/fisiologíaRESUMEN
Human S100A15 is a novel member of the S100 family of EF-hand calcium-binding proteins and was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, the protein was crystallized to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. The S100A15 protein has been cloned, expressed and purified to homogeneity and produced two crystal forms. Crystals of form I are triclinic, with unit-cell parameters a = 33.5, b = 44.3, c = 44.8 angstroms, alpha = 71.2, beta = 68.1, gamma = 67.8 degrees and an estimated two molecules in the asymmetric unit, and diffract to 1.7 angstroms resolution. Crystals of form II are monoclinic, with unit-cell parameters a = 82.1, b = 33.6, c = 52.2 angstroms, beta = 128.2 degrees and an estimated one molecule in the asymmetric unit, and diffract to 2.0 angstroms resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7.
Asunto(s)
Proteínas S100/química , Proteínas S100/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Humanos , Psoriasis/metabolismo , Proteína A7 de Unión a Calcio de la Familia S100RESUMEN
BACKGROUND: Arf GAPs are multidomain proteins that function in membrane traffic by inactivating the GTP binding protein Arf1. Numerous Arf GAPs contain a BAR domain, a protein structural element that contributes to membrane traffic by either inducing or sensing membrane curvature. We have examined the role of a putative BAR domain in the function of the Arf GAP ASAP1. RESULTS: ASAP1's N terminus, containing the putative BAR domain together with a PH domain, dimerized to form an extended structure that bound to large unilamellar vesicles containing acidic phospholipids, properties that define a BAR domain. A recombinant protein containing the BAR domain of ASAP1, together with the PH and Arf GAP domains, efficiently bent the surface of large unilamellar vesicles, resulting in the formation of tubular structures. This activity was regulated by Arf1*GTP binding to the Arf GAP domain. In vivo, the tubular structures induced by ASAP1 mutants contained epidermal growth factor receptor (EGFR) and Rab11, and ASAP1 colocalized in tubular structures with EGFR during recycling of receptor. Expression of ASAP1 accelerated EGFR trafficking and slowed cell spreading. An ASAP1 mutant lacking the BAR domain had no effect. CONCLUSIONS: The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/fisiología , Membrana Celular/ultraestructura , Receptores ErbB/metabolismo , Proteínas Activadoras de GTPasa/química , Proteínas Activadoras de GTPasa/fisiología , Factores de Ribosilacion-ADP/fisiología , Proteínas Adaptadoras Transductoras de Señales/análisis , Secuencia de Aminoácidos , Animales , Proteínas Activadoras de GTPasa/análisis , Ratones , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Transporte de Proteínas , Alineación de SecuenciaRESUMEN
Arf1 is a GTP binding protein that functions at a number of cellular sites to control membrane traffic and actin remodeling. Arf1 is regulated by site-specific GTPase-activating proteins (GAPs). The combined results of crystallographic and biochemical studies have led to the proposal that Arf1 GAPs differ in the specific interface formed with Arf1. To test this hypothesis, we have used mutagenesis to examine the interaction of three Arf GAPs (ASAP1, AGAP1, and ArfGAP1) with switch 1, switch 2, and alpha helix3 of Arf1. The GAPs were similar in being affected by mutations in switch 1 and 2. However, effects of a mutation within alpha helix3 and specific mutations within switch 1 and 2 differed among the GAPs. The largest differences were observed with a change of isoleucine 46 to aspartate ([I46D]Arf1), which reduced ASAP1-induced catalysis by approximately 10,000-fold but had a 3-fold effect on AGAP1. The reduction was due to an isolated effect on the catalytic rate, k(cat). In vivo [I46D]Arf1 had no detectable effect on the Golgi apparatus but, instead, functioned as a constitutively active mutant in the cell periphery, affecting the localization of ASAP1 and paxillin. Based on our results, we conclude that the contribution of specific residues within switch 1 of Arf to binding and achieving a transition state toward GTP hydrolysis differs among Arf GAPs.
Asunto(s)
Factor 1 de Ribosilacion-ADP/genética , Factor 1 de Ribosilacion-ADP/metabolismo , Factores de Ribosilacion-ADP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Modelos Moleculares , Animales , Catálisis , Análisis Mutacional de ADN , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Ratones , Mutación/genética , Células 3T3 NIHRESUMEN
Gene product (gp) 24 of bacteriophage T4 forms the pentameric vertices of the capsid. Using x-ray crystallography, we found the principal domain of gp24 to have a polypeptide fold similar to that of the HK97 phage capsid protein plus an additional insertion domain. Fitting gp24 monomers into a cryo-EM density map of the mature T4 capsid suggests that the insertion domain interacts with a neighboring subunit, effecting a stabilization analogous to the covalent crosslinking in the HK97 capsid. Sequence alignment and genetic data show that the folds of gp24 and the hexamer-forming capsid protein, gp23*, are similar. Accordingly, models of gp24* pentamers, gp23* hexamers, and the whole capsid were built, based on a cryo-EM image reconstruction of the capsid. Mutations in gene 23 that affect capsid shape map to the capsomer's periphery, whereas mutations that allow gp23 to substitute for gp24 at the vertices modify the interactions between monomers within capsomers. Structural data show that capsid proteins of most tailed phages, and some eukaryotic viruses, may have evolved from a common ancestor.
Asunto(s)
Proteínas de la Cápside/genética , Colifagos/metabolismo , Evolución Molecular , Modelos Moleculares , Secuencia de Aminoácidos , Proteínas de la Cápside/química , Clonación Molecular , Cristalografía por Rayos X , Datos de Secuencia Molecular , Mutación/genética , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
We have determined a 2.1 A crystal structure for human mitochondrial ClpP (hClpP), the proteolytic component of the ATP-dependent ClpXP protease. HClpP has a structure similar to that of the bacterial enzyme, with the proteolytic active sites sequestered within an aqueous chamber formed by face-to-face assembly of the two heptameric rings. The hydrophobic N-terminal peptides of the subunits are bound within the narrow (12 A) axial channel, positioned to interact with unfolded substrates translocated there by the associated ClpX chaperone. Mutation or deletion of these residues causes a drastic decrease in ClpX-mediated protein and peptide degradation. Residues 8-16 form a mobile loop that extends above the ring surface and is also required for activity. The 28 amino acid C-terminal domain, a unique feature of mammalian ClpP proteins, lies on the periphery of the ring, with its proximal portion forming a loop that extends out from the ring surface. Residues at the start of the C-terminal domain impinge on subunit interfaces within the ring and affect heptamer assembly and stability. We propose that the N-terminal peptide of ClpP is a structural component of the substrate translocation channel and may play an important functional role as well.
Asunto(s)
Endopeptidasa Clp/química , Endopeptidasa Clp/fisiología , Proteínas de Escherichia coli/química , Mitocondrias/enzimología , Mitocondrias/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Eliminación de Gen , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/química , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
Transglutaminases (TGase; protein-glutamine: amine gamma-glutamyl-transferase) are a family of calcium-dependent acyl-transfer enzymes ubiquitously expressed in mammalian cells and responsible for catalyzing covalent cross-links between proteins or peptides. A series of recent crystal structures have revealed the overall architecture of TGase enzymes, and provided a deep look at their active site, calcium and magnesium ions, and the manner by which guanine nucleotides interact with this enzyme. These structures, backed with extensive biochemical studies, are providing new insights as to how access to the enzyme's active site may be gated through the coordinated changes in cellular calcium and magnesium concentrations and GTP/GDP. Calcium-activated TGase 3 can bind, hydrolyze, and is inhibited by GTP, despite lacking structural homology with other GTP binding proteins. A structure based sequence homology among the TGase enzyme family shows that these essential structural features are shared among other members of the TGase family.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Transglutaminasas/química , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Activación Enzimática/fisiología , Guanosina Trifosfato/metabolismo , Humanos , Metales/metabolismo , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
Epidermal-type Transglutaminase 3 (TGase 3) is a Ca(2+)-dependent enzyme involved in the cross-linking of structural proteins required in the assembly of the cell envelope. We have recently shown that calcium-activated TGase 3, like TGase 2, can bind, hydrolyze, and is inhibited by GTP despite lacking structural homology with other GTP-binding proteins. Here we report the crystal structure determined at 2.0 A resolution of TGase 3 in complex with GMP to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) (0.4 microm), GTP (0.6 microm), GDP (1.0 microm), GMP (0.4 microm), and ATP (28.0 microm). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTPgammaS and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest trade mark data base (Tripos Inc.) based on superimposition of GTPgammaS, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (K(d) = 4.1 microm), TP395289 (K(d) = 38.5 microm), TP394305 (K(d) = 1.0 mm). Remarkably, these compounds do not inhibit but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches.
Asunto(s)
Guanosina Monofosfato/química , Adenosina Trifosfato/química , Algoritmos , Compuestos de Boro/farmacología , Membrana Celular/metabolismo , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Electrones , Guanina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Humanos , Hidrólisis , Cinética , Ligandos , Modelos Químicos , Modelos Moleculares , Fosfatos/química , Unión Proteica , Conformación Proteica , Espectrometría de FluorescenciaRESUMEN
Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.
Asunto(s)
Proteínas de Unión al Calcio/biosíntesis , Proteínas de Unión al Calcio/genética , Calcio/química , Regulación Enzimológica de la Expresión Génica , Magnesio/química , Transglutaminasas/biosíntesis , Transglutaminasas/genética , Secuencia de Aminoácidos , Sitios de Unión , Calorimetría , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Genoma , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Humanos , Hidrólisis , Iones , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Isoformas de Proteínas , Proteínas Recombinantes/química , Transducción de Señal , Especificidad por Sustrato , Termodinámica , Factores de TiempoRESUMEN
Transglutaminase enzymes (TGases) catalyze the calcium dependent formation of an isopeptide bond between protein-bound glutamine and lysine substrates. Previously we have shown that activated TGase 3 acquires two additional calcium ions at site two and three. The calcium ion at site three results in the opening of a channel. At this site, the channel opening and closing could modulate, depending on which metal is bound. Here we propose that the front of the channel could be used by the two substrates for enzyme reaction. We propose that the glutamine substrate is directed from Trp236 into the enzyme, shown by molecular docking. Then a lysine substrate approaches the opened active site to engage Trp327, leading to formation of the isopeptide bond. Further, direct comparisons of the structures of TGase 3 with other TGases have allowed us to identify several residues that might potentially be involved in generic and specific recognition of the glutamine and lysine substrates.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Transglutaminasas/metabolismo , Animales , Sitios de Unión , Calcio/metabolismo , Glutamina/metabolismo , Humanos , Lisina/metabolismo , Modelos Químicos , Modelos Moleculares , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
The transglutaminase 3 enzyme is widely expressed in many tissues including epithelia. We have shown previously that it can bind three Ca2+ ions, which in site one is constitutively bound, while those in sites two and three are acquired during activation and are required for activity. In particular, binding at site three opens a channel through the enzyme and exposes two tryptophan residues near the active site that are thought to be important for enzyme reaction. In this study, we have solved the structures of three more forms of this enzyme by x-ray crystallography in the presence of Ca2+ and/or Mg2+, which provide new insights on the precise contribution of each Ca2+ ion to activation and activity. First, we found that Ca2+ ion in site one can be exchanged with difficulty, and it has a binding affinity of Kd = 0.3 microm (DeltaH = -6.70 +/- 0.52 kcal/mol), which suggests it is important for the stabilization of the enzyme. Site two can be occupied by some lanthanides but only Ca2+ of the Group 2 family of alkali earth metals, and its occupancy are required for activity. Site three can be occupied by some lanthanides, Ca2+,or Mg2+; however, when Mg2+ is present, the enzyme is inactive, and the channel is closed. Thus Ca2+ binding in both sites two and three cooperate in opening the channel. We speculate that manipulation of the channel opening could be controlled by intracellular cation levels. Together, these data have important implications for reaction mechanism of the enzyme: the opening of a channel perhaps controls access to and manipulation of substrates at the active site.