RESUMEN
A series of N-tert-butylacetyl-l-tert-butylglycyl-l-Ngamma, Ngamma-dimethylasparagyl-l-alanyl-derived inhibitors (trifluoromethyl ketone 1, pentafluoroethyl ketone, 2, methyl ketone 3, and alpha-ketoamide 4, with respective KI values of 1.1, 0.1, 2100, and 0.2 microM) of the human cytomegalovirus protease were used to study the effect of binding of peptidyl inhibitors on the intrinsic fluorescence and CD properties of the enzyme. In the presence of saturating concentrations of compounds 1, 2, and 4, an identical blue shift in the fluorescence maximum of the enzyme upon specific tryptophan excitation was observed relative to that of the free protease. In the case of the methyl ketone 3, whose inhibition of the enzyme does not involve formation of a covalent adduct as evidenced by 13C NMR studies of carbonyl-labeled inhibitors, the blue shift in the emission was also observed. For both compounds 1 and 2 which exhibit slow-binding kinetics, the observed rate constants for the slow onset of inhibition of substrate hydrolysis correlate well with the kobs values of the time-dependent change in the emission spectra. Studies employing a double mutant of HCMV protease Ala143Gln/Trp42Phe identified Trp-42 as the principal fluorescence reporter. Taken together with information provided by our recent elucidation of the crystallographic structure of the enzyme [Tong, L., Qian, C., Massariol, M.-J., Bonneau, P. R., Cordingley, M. G., & Lagacé, L. (1996) Nature 383, 272], these observations are consistent with the inhibition of HCMV protease by peptidyl ketones involving a conformational change of the protease. A mechanism involving a kon limited by dehydration of the hydrated species, followed by rapid ligand binding and a conformational change prior to covalent adduct formation, is proposed for activated inhibitors such as 1 and 2.
Asunto(s)
Citomegalovirus/química , Endopeptidasas/química , Inhibidores de Proteasas/química , Conformación Proteica , Serina Endopeptidasas , Citomegalovirus/enzimología , Endopeptidasas/metabolismo , Humanos , Cetonas/química , Cetonas/metabolismo , Cetonas/farmacología , Espectroscopía de Resonancia Magnética , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacologíaRESUMEN
One of the key steps involved in T-cell activation is binding of the tyrosine kinase ZAP-70 via its two SH2 domains to peptide segments termed tyrosine-based activation motifs (ITAM) which are present in three of the T-cell receptor (TCR) subunits. The crystal structure of the ZAP-70 SH2 domains complexed to phosphopeptide revealed that the amino-terminal phosphotyrosine-binding pocket is formed at the interface between the two SH2 domains. This study was designed to further characterize the binding between TCR zeta ITAM1 and the ZAP-70 SH2 domains as well as to assess the change in conformation of SH2 domain structure upon zeta ITAM1 binding. BIAcore analysis of wild type and nonfunctional single-point mutants of ZAP-70 SH2 domains demonstrated that the amino-terminal SH2 domain can bind phosphopeptide in the absence of a functional carboxyl-terminal SH2 domain. In addition, the amino-terminal SH2 domain prefers the RREEpYDVLDK sequence of zeta chain ITAM1 over the GQNQLpYNELNL sequence. To assess changes in protein conformation upon ITAM binding to ZAP-70 SH2 domains, fluorescence spectroscopy and analytical ultracentrifugation experiments were performed. A significant blue shift in the tryptophan emission spectrum of the SH2 domains was observed in the presence of saturating amounts of phosphopeptide, indicating a loss in solvent exposure for the tryptophan residues in the protein-phosphopeptide complex. This was accompanied by changes in the frictional coefficient consistent with a compacting of the protein structure. Finally, thermal denaturation experiments showed an increase in stability and cooperativity in unfolding for the protein-phosphopeptide complex relative to the protein alone.
Asunto(s)
Proteínas de la Membrana/genética , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos T/genética , Secuencia de Aminoácidos , Sitios de Unión , Clonación Molecular , Vectores Genéticos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Conformación Proteica , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70RESUMEN
Nevirapine is a highly potent and specific inhibitor of human immunodeficiency virus type 1 (HIV-1) polymerase, but is inactive against HIV-2 and other polymerase. Previous studies demonstrated that residues 176-190 of HIV-1 reverse transcriptase (RT) can confer nevirapine sensitivity to HIV-2 RT. To better characterize the role of this sequence in HIV-1 RT, we have progressively substituted residues 176-190 of HIV-2 RT for those of HIV-1 RT and monitored the impact on the kinetic properties; inhibitory activity of nevirapine (11-cyclopropyl-5,11-dihydro-4-methyl-6H-dipyrido[2,3-b:2',3'-e] [1,4]diazepin-6-one), E-BPU (5-ethyl-1-benzyloxymethyl-6-(phenylthio)-uracil), and TIBO-R82150 ((+)-S-4,5,6,7-tetrahydro-5-methyl-6-(3-methyl-2-butenyl)imidazo[4,5,1-j k] [1,4]benzodiazepin-2(1H)-thione); and inhibitor-induced fluorescence changes of the mutant enzymes. The study revealed that in addition to Try-181 and Tyr-188, a new amino acid residue (Gly-190) plays an important role in determining susceptibility to nevirapine and E-BPU, but not to TIBO-R82150. These data argue that these non-nucleoside inhibitors fit differently, even though they share a common binding pocket. Nevirapine was seen to exert inhibitory activity by altering the interaction of the enzyme with the template-primer. Kinetic parameters were modulated by the template (DNA versus RNA) as well as by some of the mutations.
Asunto(s)
Antivirales/farmacología , VIH-1/enzimología , VIH-2/enzimología , Piridinas/farmacología , ADN Polimerasa Dirigida por ARN/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Transcriptasa Inversa del VIH , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Nevirapina , Inhibidores de la Transcriptasa Inversa , Espectrometría de FluorescenciaRESUMEN
Predictive models of soil and plant processes should be of much benefit both in developing and developed countries. They can assist in enabling agronomic practices to be better adjusted for differences in conditions; in avoiding disasters that can accompany change in land use; in minimizing waste and environmental pollution, and in modifying and implementing legislation. These views are discussed in the light of recent advances. Particular attention is drawn to (i) the excellent relationships linking average national yields, nutrient uptakes, etc. to single factors such as average fertilizer application; (ii) equations for predicting the behaviour of added chemicals and water in soil that have been obtained by rigorous deduction from physical and chemical laws; (iii) the discovery of semi-empirical, but nevertheless widely applicable quantitative relations for key soil and plant processes, and (iv) the formulation and use of computer models for field situations. There is a pressing need to find ways of presenting the outcome of this work so that it can be more widely applied in practice.