RESUMEN
BACKGROUND: The lymphocyte-specific kinase Lck is a member of the Src family of non-receptor tyrosine kinases. Lck catalyzes the initial phosphorylation of T-cell receptor components that is necessary for signal transduction and T-cell activation. On the basis of both biochemical and genetic studies, Lck is considered an attractive cell-specific target for the design of novel T-cell immunosuppressants. To date, the lack of detailed structural information on the mode of inhibitor binding to Lck has limited the discovery of novel Lck inhibitors. RESULTS: We report here the high-resolution crystal structures of an activated Lck kinase domain in complex with three structurally distinct ATP-competitive inhibitors: AMP-PNP (a non-selective, non-hydrolyzable ATP analog); staurosporine (a potent but non-selective protein kinase inhibitor); and PP2 (a potent Src family selective protein tyrosine kinase inhibitor). Comparison of these structures reveals subtle but important structural changes at the ATP-binding site. Furthermore, PP2 is found to access a deep, hydrophobic pocket near the ATP-binding cleft of the enzyme; this binding pocket is not occupied by either AMP-PNP or staurosporine. CONCLUSIONS: The potency of staurosporine against Lck derives in part from an induced movement of the glycine-rich loop of the enzyme upon binding of this ligand, which maximizes the van der Waals interactions present in the complex. In contrast, PP2 binds tightly and selectively to Lck and other Src family kinases by making additional contacts in a deep, hydrophobic pocket adjacent to the ATP-binding site; the amino acid composition of this pocket is unique to Src family kinases. The structures of these Lck complexes offer useful structural insights as they demonstrate that kinase selectivity can be achieved with small-molecule inhibitors that exploit subtle topological differences among protein kinases.
Asunto(s)
Inhibidores Enzimáticos/química , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/química , Linfocitos/enzimología , Familia-src Quinasas/antagonistas & inhibidores , Adenosina Trifosfato/química , Adenilil Imidodifosfato/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fosfotirosina/metabolismo , Unión Proteica , Piridinas/química , Alineación de Secuencia , Estaurosporina/químicaRESUMEN
BACKGROUND: Hepatitis C virus (HCV) represents a major health concern as it is responsible for a significant number of hepatitis cases worldwide. Much research has focused on the replicative enzymes of HCV as possible targets for more effective therapeutic agents. HCV NS3 helicase may provide one such suitable target. Helicases are enzymes which can unwind double-stranded regions of DNA or RNA in an ATP-dependent reaction. The structures of several helicases have been published but the structural details as to how ATP binding and hydrolysis are coupled to RNA unwinding are unknown. RESULTS: The structure of the HCV NS3 RNA helicase domain complexed with a single-stranded DNA oligonucleotide has been solved to 2.2 A resolution. The protein consists of three structural domains with the oligonucleotide lying in a groove between the first two domains and the third. The first two domains have an adenylate kinase like fold, including a phosphate-binding loop in the first domain. CONCLUSIONS: HCV NS3 helicase is a member of a superfamily of helicases, termed superfamily II. Residues of NS3 helicase which are conserved among superfamily II helicases line an interdomain cleft between the first two domains. The oligonucleotide binds in an orthogonal binding site and contacts relatively few conserved residues. There are no strong sequence-specific interactions with the oligonucleotide bases.
Asunto(s)
ADN de Cadena Simple/química , Hepacivirus/enzimología , Secuencia de Aminoácidos , Secuencia Conservada/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas de Unión al ARN/fisiología , Alineación de Secuencia , Proteínas no Estructurales Virales , Proteínas Virales/químicaRESUMEN
Infection by hepatitis C viruses (HCVs) is a serious medical problem with no broadly effective treatment available for the progression of chronic hepatitis. The catalytic activity of a viral serine protease located in the N-terminal one-third of nonstructural protein 3 (NS3) is required for polyprotein processing at four site-specific junctions. The three-dimensional crystal structure of the NS3-NS4A co-complex [Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., O'Malley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R., & Thomson, J. A. (1996) Cell 87, 343-355] delineates a small hydrophobic region within the 54-residue NS4A protein that intercalates with and makes extensive contacts to the core of the protease. The current investigation addresses the mechanism of NS3 protease catalytic activation by NS4A utilizing a small synthetic NS4A peptide (residues 1678-1691 of the virus polyprotein sequence) and the recombinantly expressed protease domain of NS3. The addition of NS4A dramatically increased NS3 kcat and kcat/Km catalytic parameters when measured against small peptide substrates representing the different site-specific junctions of the polyprotein. The catalytic effect of natural and non-natural amino acid substitutions at the P1 position in a 5A/5B peptide substrate was investigated. NS3-NS4A demonstrated a marked catalytic preference for the cysteine residue commonly found in authentic substrates. The pH dependence of the NS3 hydrolysis reaction is not affected by the presence of NS4A. This result suggests that NS4A does not change the pKa values of the active site residues of NS3 protease. A steady state kinetic analysis was performed and indicated that the binding of NS4A and the peptide substrate occurs in an ordered fashion during the catalytic cycle, with NS4A binding first. Two distinct kinetic classes of peptidyl inhibitors based upon the 5A/5B cleavage site were identified. An NS4A-independent class is devoid of prime residues. A second class of inhibitors is NS4A-dependent and contains a natural or non-natural cyclic amino acid substituted for the commonly found P1' residue serine. These inhibitors display an up to 80-fold increase in affinity for NS3 protease in the presence of NS4A. Sequential truncation of prime and P residues from this inhibitor class demonstrated the fact that the P4' and P1' residues are crucial for potent inhibition. The selectivity of this NS4A effect is interpreted using a model of the 5A/5B decapeptide substrate bound to the active site of the NS3-NS4A structure.
Asunto(s)
Serina Endopeptidasas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Secuencia de Aminoácidos , Cinética , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Serina Endopeptidasas/química , Relación Estructura-Actividad , Especificidad por Sustrato , Proteínas no Estructurales Virales/químicaRESUMEN
The hepatitis C virus (HCV) nonstructural 3 protein (NS3) is a 70-kDa multifunctional enzyme with three known catalytic activities segregated in two somewhat independent domains. The essential machinery of a serine protease is localized in the N-terminal one-third of the protein, and nucleoside triphosphatase (NTPase) and helicase activities reside in the remaining C-terminal region. NS4A is a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, and a central stretch of hydrophobic residues in NS4A form an integral structural component of the NS3 serine protease domain. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic processing in vivo. This may reflect economical packaging of essential viral replicative components, but it could also mean that there is functional interdependence between the two domains. In this study, a full-length NS3-NS4A complex was isolated after expression and autoprocessing in transiently transfected COS cells. The protein was used to examine the effects of polynucleotides on the NTPase, helicase, and protease activities. Unlike the previously reported behavior of a separately expressed NS3 helicase domain, the full NS3-NS4A complex demonstrated optimal NTPase activity between pH 7.5 and 8.5. All three NS3-NS4A activities were modulated by polynucleotides, with poly(U) having the most remarkable effect. These findings suggest that the domains within NS3 may influence the activity of one another and that the interplay of HCV genomic elements may regulate the enzyme activities of this complex HCV replicase component.
Asunto(s)
Ácido Anhídrido Hidrolasas/efectos de los fármacos , ADN Helicasas/efectos de los fármacos , Endopeptidasas/efectos de los fármacos , Polinucleótidos/farmacología , Proteínas no Estructurales Virales/efectos de los fármacos , Animales , Células COS , Nucleósido-Trifosfatasa , Poli U/farmacología , TransfecciónRESUMEN
An estimated 1% of the global human population is infected by hepatitis C viruses (HCVs), and there are no broadly effective treatments for the debilitating progression of chronic hepatitis C. A serine protease located within the HCV NS3 protein processes the viral polyprotein at four specific sites and is considered essential for replication. Thus, it emerges as an attractive target for drug design. We report here the 2.5 angstrom resolution X-ray crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator peptide. The protease has a chymotrypsin-like fold and features a tetrahedrally coordinated metal ion distal to the active site. The NS4A peptide intercalates within a beta sheet of the enzyme core.
Asunto(s)
Hepacivirus/enzimología , Proteínas no Estructurales Virales/ultraestructura , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Alineación de Secuencia , Especificidad por Sustrato , Proteínas no Estructurales Virales/metabolismo , ZincRESUMEN
To define the presence and potential role of platelet-associated protease inhibitors, we initiated a study designed to characterize the platelet components that are responsible for the formation of two SDS-stable complexes of approximately 58 and 70 kDa initially observed following the incubation of 125I-thrombin and human platelets. We demonstrate that thermal-mediated unfolding of the 58-kDa complex between 125I-thrombin and a nonsecreted platelet protein leads to an apparent molecular mass of 70 kDa. This platelet component is functionally and immunologically indistinguishable from the cytoplasmic antiproteinase (CAP), also known as placental thrombin inhibitor, a recently cloned member of the ovalbumin family of intracellular serpins (serine proteinase inhibitors). CAP-specific mRNA and antigen were detected in human platelets, suggesting that CAP synthesis occurs concurrent with platelet development. Utilizing quantitative immunoblotting, CAP antigen was estimated at 1.014 +/- 0.181 microg/10(9) nonstimulated platelets. After platelet activation with the calcium ionophore A23187, CAP antigen was detected in released microparticles at approximately 0. 195 +/- 0.031 microg/10(9) platelets and a fraction of platelet CAP was proteolytically modified. We provide evidence that these lower molecular mass species arise by cleavage of CAP at or near the reactive site loop. Most importantly, molecular sieving chromatography indicates the presence of an approximately 68-kDa SDS-labile complex between cleaved CAP and a cellular component in A23187-stimulated platelets, suggesting a physiological target of this intracellular serpin and a potential role for this inhibitor in regulating proteolytic activity that may be formed during platelet activation.
Asunto(s)
Plaquetas/metabolismo , Proteínas Sanguíneas/metabolismo , Proteínas/metabolismo , Secuencia de Bases , Proteínas Sanguíneas/efectos de los fármacos , Western Blotting , Calcimicina/farmacología , Cartilla de ADN , Hidrólisis , Péptidos y Proteínas de Señalización Intracelular , Radioisótopos de Yodo , Datos de Secuencia Molecular , Activación Plaquetaria , Dodecil Sulfato de Sodio , Trombina/metabolismoRESUMEN
A human placental lambda gt11 cDNA library was screened for sequences encoding proteins related to human proteinase inhibitor 6 (PI6), and two plaques were identified that displayed weak hybridization at high stringency. Isolation and characterization of the DNA inserts revealed two novel sequences encoding proteins composed of 376 and 374 amino acids with predicted molecular masses of approximately 42 kDa. The novel proteins displayed all of the structural features unique to the ovalbumin family of intracellular serpins including the apparent absence of a cleavable N-terminal signal sequence. The degree of amino acid sequence identity between the novel serpins and PI6 (63-68%) significantly exceeds that of any other combination of known intracellular serpins. The two novel serpins encoded by the two novel cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and proteinase inhibitor 9 (PI9). The putative reactive center P1-P1' residues for PI8 and PI9 were identified as Arg339-Cys340 and Glu340-Cys341, respectively. PI9 appears to be unique in that it is the first human serpin identified with an acidic residue in the reactive center P1 position. In addition, the reactive center loop of PI9 exhibits 54% identity with residues found in the reactive center loop of the cowpox virus CrmA serpin. Two PI8 transcripts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern analysis in equal and greatest abundance in liver and lung, while the 1.4-kb mRNA was in excess over the 3.8-kb mRNA in skeletal muscle and heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and greatest abundance in lung and placenta and were weakly detected in all other tissues. PI8 and PI9 were expressed in baby hamster kidney and yeast cells, respectively. Immunoblot analyses using rabbit anti-PI6 IgG indicated the presence of PI8 in the cytosolic fraction of stably transfected cells that formed an SDS-stable 67-kDa complex with human thrombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin-agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 42 kDa. Purified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu-C and did not form an SDS-stable complex when incubated with thrombin. The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.
Asunto(s)
Ovalbúmina/biosíntesis , Serpinas/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cricetinae , ADN/química , Femenino , Biblioteca de Genes , Humanos , Riñón , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Insercional , Ovalbúmina/química , Ovalbúmina/genética , Placenta/metabolismo , Embarazo , Conejos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Saccharomyces cerevisiae , Homología de Secuencia de Aminoácido , Serpinas/química , Serpinas/genética , TransfecciónRESUMEN
The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.
Asunto(s)
Clonación Molecular , ADN Complementario/genética , Factor Xa/metabolismo , Proteínas/genética , Inhibidores de Serina Proteinasa/genética , Tripsina/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Codón , ADN Complementario/química , Femenino , Glicosilación , Humanos , Péptidos y Proteínas de Señalización Intracelular , Cinética , Datos de Secuencia Molecular , Especificidad de Órganos , Placenta/química , Proteínas/química , Proteínas/farmacología , ARN Mensajero/análisis , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacología , Clorometilcetona de Tosilfenilalanila/metabolismoRESUMEN
A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.