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1.
Biochem Biophys Res Commun ; 482(4): 1289-1295, 2017 Jan 22.
Artículo en Inglés | MEDLINE | ID: mdl-27993680

RESUMEN

Human Protein tyrosine kinase 6 (PTK6)(EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed five-fold or more in human breast tumors and breast cancer cell lines but its expression being low or completely absent from normal mammary gland. There is a recent interest in targeting PTK6-positive breast cancer by developing small molecule inhibitor against PTK6. Novel imidazo[1,2-a]pyrazin-8-amines (IPA) derivative compounds and FDA approved drug, Dasatinib are reported to inhibit PTK6 kinase activity with IC50 in nM range. To understand binding mode of these compounds and key interactions that drive the potency against PTK6, one of the IPA compounds and Dasatinib were chosen to study through X-ray crystallography. The recombinant PTK6 kinase domain was purified and co-crystallized at room temperature by the sitting-drop vapor diffusion method, collected X-ray diffraction data at in-house and resolved co-crystal structure of PTK6-KD with Dasatinib at 2.24 Å and with IPA compound at 1.70 Å resolution. Both these structures are in DFG-in & αC-helix-out conformation with unambiguous electron density for Dasatinib or IPA compound bound at the ATP-binding pocket. Relative difference in potency between Dasatinib and IPA compound is delineated through the additional interactions derived from the occupation of additional pocket by Dasatinib at gatekeeper area. Refined crystallographic coordinates for the kinase domain of PTK6 in complex with IPA compound and Dasatinib have been submitted to Protein Data Bank under the accession number 5DA3 and 5H2U respectively.


Asunto(s)
Aminas/química , Neoplasias de la Mama/tratamiento farmacológico , Proteínas de Neoplasias/química , Proteínas Tirosina Quinasas/química , Adenosina Trifosfato/química , Neoplasias de la Mama/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Dasatinib/química , Difusión , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Imidazoles/química , Concentración 50 Inhibidora , Unión Proteica , Dominios Proteicos , Mapeo de Interacción de Proteínas
2.
Biochem Biophys Res Commun ; 478(2): 637-42, 2016 09 16.
Artículo en Inglés | MEDLINE | ID: mdl-27480927

RESUMEN

Human Protein tyrosine kinase 6 (PTK6) (EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed in a majority of human breast tumors and breast cancer cell lines, but its expression is low or completely absent in normal mammary glands. In the recent past, several studies have suggested that PTK6 is a potential therapeutic target in cancer. To understand its structural and functional properties, the PTK6 kinase domain (PTK6-KD) gene was cloned, overexpressed in a baculo-insect cell system, purified and crystallized at room temperature. X-ray diffraction data to 2.33 Å resolution was collected on a single PTK6-KD crystal, which belonged to the triclinic space group P1. The Matthews coefficient calculation suggested the presence of four protein molecules per asymmetric unit, with a solvent content of ∼50%.The structure has been solved by molecular replacement and crystal structure data submitted to the protein data bank under the accession number 5D7V. This is the first report of apo PTK6-KD structure crystallized in DFG-in and αC-helix-out conformation.


Asunto(s)
Mutación , Proteínas de Neoplasias/química , Proteínas Tirosina Quinasas/química , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Pruebas de Enzimas , Expresión Génica , Humanos , Cinética , Modelos Moleculares , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Conformación Proteica en Hélice alfa , Dominios Proteicos , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Células Sf9 , Spodoptera , Relación Estructura-Actividad
3.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 9): 1717-25, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23999295

RESUMEN

XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.


Asunto(s)
Caspasa 3/química , Caspasa 3/metabolismo , Inhibidores de Caspasas/química , Proteínas Inhibidoras de la Apoptosis/química , Nucleopoliedrovirus/química , Proteínas Virales/química , Proteína Inhibidora de la Apoptosis Ligada a X/química , Secuencia de Aminoácidos , Apoproteínas/química , Apoproteínas/genética , Apoptosis/genética , Cristalografía por Rayos X , Humanos , Proteínas Inhibidoras de la Apoptosis/genética , Datos de Secuencia Molecular , Familia de Multigenes/genética , Nucleopoliedrovirus/genética , Oligopéptidos/química , Oligopéptidos/genética , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína/genética , Proteínas Virales/genética , Proteína Inhibidora de la Apoptosis Ligada a X/genética
4.
Biochemistry ; 47(20): 5608-15, 2008 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-18442260

RESUMEN

Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-L-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [ N-formimino-L-glutamate (NIG)] and an inhibitor [3-(2,5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.


Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/metabolismo , Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/clasificación , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Secuencia Conservada , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Histidina/química , Histidina/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia
5.
BMC Struct Biol ; 7: 62, 2007 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-17908300

RESUMEN

BACKGROUND: Pfam is a comprehensive collection of protein domains and families, with a range of well-established information including genome annotation. Pfam has two large series of functionally uncharacterized families, known as Domains of Unknown Function (DUFs) and Uncharacterized Protein Families (UPFs). RESULTS: Crystal structures of two proteins from Deinococcus radiodurans and Streptomyces coelicolor belonging to Pfam protein family DUF178 (ID: PF02621) have been determined using Selenium-Single-wavelength Anomalous Dispersion (Se-SAD). Based on the structure, we have identified the putative function for this family of protein. CONCLUSION: Unexpectedly, we found that DUF178 Pfam is remarkably similar to Pfam family DUF191 suggesting that the sequence-based classification alone may not be sufficient to classify proteins into Pfam families.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Deinococcus/química , Streptomyces coelicolor/química , Homología Estructural de Proteína , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Ligandos , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido
6.
Protein Sci ; 14(12): 3089-100, 2005 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-16322583

RESUMEN

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branched-chain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 A resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed alpha/beta structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly alpha-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunit-to-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.


Asunto(s)
Escherichia coli/enzimología , Evolución Molecular , Cetoácido Reductoisomerasa/química , Secuencia de Aminoácidos , Sitios de Unión , Calcio/química , Cationes Bivalentes/química , Cristalografía por Rayos X , Dimerización , Escherichia coli/genética , Cetoácido Reductoisomerasa/clasificación , Cetoácido Reductoisomerasa/genética , Cetoácido Reductoisomerasa/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/enzimología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Spinacia oleracea/enzimología , Homología Estructural de Proteína
7.
FEBS J ; 272(2): 593-602, 2005 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-15654896

RESUMEN

Ketol-acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched-chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH-dependent reduction of a 2-ketoacid. The 2-ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2-ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2-ketoacid and provides a rationale for the requirement for NADPH by the isomerase.


Asunto(s)
Oxidorreductasas de Alcohol/metabolismo , Oxidorreductasas de Alcohol/química , Sitios de Unión , Catálisis , Cetoácido Reductoisomerasa , Cinética , Magnesio/metabolismo , Mutagénesis Sitio-Dirigida , NADP/metabolismo
8.
BMC Biotechnol ; 4: 2, 2004 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-15070414

RESUMEN

BACKGROUND: Site-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR. RESULTS: The strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants. CONCLUSION: This strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.


Asunto(s)
Cartilla de ADN , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa/métodos , Oxidorreductasas de Alcohol/genética , ADN/aislamiento & purificación , Electroforesis en Gel de Agar , Cetoácido Reductoisomerasa
9.
Indian J Endocrinol Metab ; 17(Suppl 1): S157-9, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24251142

RESUMEN

Pulse methylprednisolone therapy is the recommended therapy for moderate to severe and active ophthalmopathy, but high dose pulse methylprednisolone therapy is marred by the chances of fulminant hepatic failure and the high cost of therapy. Dexamethasone pulse therapy can be considered as an alternative to pulse methylprednisolone therapy. A prospective randomized control trial was carried out in 21 patients comparing pulse dexamethasone therapy versus pulse methyprednisolone therapy in Graves's ophthalmopathy. This study proved that pulse dexamethasone therapy is a cheaper and equally effective therapy for Graves's ophthalmopathy and the cost of therapy is reduced to at least 1/8(th) s. Furthermore, dexa had a better effect on reduction of exophthalmos. The dreaded complication of fulminant hepatic failure, associated with high dose of methylprednisolone, is not seen with dexa therapy.

10.
Indian J Endocrinol Metab ; 17(Suppl 1): S283-5, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24251187

RESUMEN

Mauriac syndrome is a rare syndrome associated with type 1 diabetes (T1DM) in children presenting with growth retardation, hepatomegaly, and cushingoid features. Recently, there has been re-emergence of this syndrome, especially with the use of premix insulin. A 15-year old type 1 diabetic boy, who was on premix insulin with erratic blood glucose, was referred to us for evaluation of short stature. He had significant short stature, hepatomegaly, and cushingoid features. His growth hormone (GH) stimulation was normal, and so was the overnight dexamethasone suppression test, based on which the diagnosis of Mauriac syndrome was reported. He was made to switch over to basal bolus regime, and was advised to follow-up for 6 months. He had reduction in hepatomegaly and a height gain of 3 cms.

12.
Acta Crystallogr D Biol Crystallogr ; 60(Pt 8): 1432-4, 2004 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15272168

RESUMEN

Ketol-acid reductoisomerase (EC 1.1.1.86) catalyses the second reaction in the biosynthesis of branched-chain amino acids. The reaction involves an Mg2+ -dependent alkyl migration followed by an NADPH-dependent reduction of the 2-keto group. Here, the crystallization of the Escherichia coli enzyme is reported. A form with a C-terminal hexahistidine tag could be crystallized under 18 different conditions in the absence of NADPH or Mg2+ and a further six crystallization conditions were identified with one or both ligands. With the hexahistidine tag on the N-terminus, 20 crystallization conditions were found, some of which required the presence of NADPH, NADP+, Mg2+ or a combination of ligands. Finally, the selenomethionine-substituted enzyme with the N-terminal tag crystallized under 15 conditions. Thus, the enzyme is remarkably easy to crystallize. Most of the crystals diffract poorly but several data sets were collected at better than 3.2 A resolution; attempts to phase them are currently in progress.


Asunto(s)
Oxidorreductasas de Alcohol/química , Escherichia coli/enzimología , Oxidorreductasas de Alcohol/biosíntesis , Oxidorreductasas de Alcohol/genética , Oxidorreductasas de Alcohol/aislamiento & purificación , Cristalización , Cristalografía por Rayos X , Escherichia coli/genética , Expresión Génica , Cetoácido Reductoisomerasa
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