RESUMEN
The mature capsids of HIV and other retroviruses organize and package the viral genome and its associated enzymes for delivery into host cells. The HIV capsid is a fullerene cone: a variably curved, closed shell composed of approximately 250 hexamers and exactly 12 pentamers of the viral CA protein. We devised methods for isolating soluble, assembly-competent CA hexamers and derived four crystallographically independent models that define the structure of this capsid assembly unit at atomic resolution. A ring of six CA N-terminal domains form an apparently rigid core, surrounded by an outer ring of C-terminal domains. Mobility of the outer ring appears to be an underlying mechanism for generating the variably curved lattice in authentic capsids. Hexamer-stabilizing interfaces are highly hydrated, and this property may be key to the formation of quasi-equivalent interactions within hexamers and pentamers. The structures also clarify the molecular basis for capsid assembly inhibition and should facilitate structure-based drug design strategies.
Asunto(s)
Proteínas de la Cápside/química , VIH-1/química , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , VIH-1/metabolismo , Modelos Moleculares , Polímeros/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Knowledge of the role of conserved residues in the ligand channel of heme-copper oxidases is critical for understanding how the protein scaffold modulates the function of these enzymes. In this study, we investigated the role of the conserved valine 236 in the ligand channel of ba3 cytochrome c oxidase from Thermus thermophilus by mutating the residue to a more polar (V236T), smaller (V236A), or larger (V236I, V236N, V236L, V236M, and V236F) residue. The crystal structures of the mutants were determined, and the effects of the mutations on the rates of CO, O2, and NO binding were investigated. O2 reduction and NO binding were unaffected in V236T, while the oxidation of heme b during O-O bond cleavage was not detected in V236A. The V236A results are attributed to a decrease in the rate of electron transfer between heme b and heme a3 during O-O bond cleavage in V236A, followed by faster re-reduction of heme b by CuA. This interpretation is supported by classical molecular dynamics simulations of diffusion of O2 to the active site in V236A that indicated a larger distance between the two hemes compared to that in the wild type and increased contact of heme a3 with water and weakened interactions with residues R444 and R445. As the size of the mutant side chain increased and protruded more into the ligand cavity, the rates of ligand binding decreased correspondingly. These results demonstrate the importance of V236 in facilitating access of ligands to the active site in T. thermophilus ba3.
Asunto(s)
Proteínas Bacterianas/metabolismo , Grupo Citocromo b/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Thermus thermophilus/enzimología , Valina/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Dominio Catalítico , Cristalización , Cristalografía por Rayos X , Grupo Citocromo b/química , Grupo Citocromo b/genética , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/genética , Hemo/química , Hemo/metabolismo , Cinética , Ligandos , Simulación de Dinámica Molecular , Mutación Missense , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismo , Unión Proteica , Dominios Proteicos , Espectrofotometría , Thermus thermophilus/genética , Valina/química , Valina/genéticaRESUMEN
Using a combined structural and biochemical approach, the functional importance of a recently described peripheral pocket bounded by the E-, F-, G-, and I-helices in CYP2B4 and 2B6 was probed. Three series of 4-substituted-7-alkoxycoumarin derivatives with -H, -CH3, or -CF3 at the 4 position of the coumarin core were used initially to monitor functional differences between CYP2B4 and 2B6. 7-Ethoxy-4-(trifluoromethyl)coumarin (7-EFC) displayed the highest catalytic efficiency among these substrates. Mutants were made to alter side-chain polarity (V/E194Q) or bulk (F/Y244W) to alter access to the peripheral pocket. Modest increases in catalytic efficiency of 7-EFC O-deethylation by the mutants were magnified considerably by chlorination or bromination of the substrate ethoxy chain. A structure of CYP2B6 Y244W in complex with (+)-α-pinene was solved at 2.2 Å and showed no CYMAL-5 in the peripheral pocket. A ligand free structure of CYP2B4 F244W was solved at 3.0 Å with CYMAL-5 in the peripheral pocket. In both instances, comparison of the respective wild-type and mutant CYP2B enzymes revealed that CYMAL-5 occupancy of the peripheral pocket had little effect on the topology of active site residue side-chains, despite the fact that the peripheral pocket and active site are located on opposite sides of the I-helix. Analysis of available CYP2B structures suggest that the effect of the amino acid substitutions within the peripheral pocket derive from altered interactions between the F and G helices.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Cumarinas/metabolismo , Citocromo P-450 CYP2B6/metabolismo , Modelos Moleculares , Alquilación , Sustitución de Aminoácidos , Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Sitios de Unión , Dominio Catalítico , Cumarinas/química , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/genética , Familia 2 del Citocromo P450 , Halogenación , Humanos , Cinética , Ligandos , Conformación Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Mutación , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Crystal structures of CYP2B35 and CYP2B37 from the desert woodrat were solved in complex with 4-(4-chlorophenyl)imidazole (4-CPI). The closed conformation of CYP2B35 contained two molecules of 4-CPI within the active site, whereas the CYP2B37 structure demonstrated an open conformation with three 4-CPI molecules, one within the active site and the other two in the substrate access channel. To probe structure-function relationships of CYP2B35, CYP2B37, and the related CYP2B36, we tested the O-dealkylation of three series of related substrates-namely, 7-alkoxycoumarins, 7-alkoxy-4-(trifluoromethyl)coumarins, and 7-alkoxy-4-methylcoumarins-with a C1-C7 side chain. CYP2B35 showed the highest catalytic efficiency (kcat/KM) with 7-heptoxycoumarin as a substrate, followed by 7-hexoxycoumarin. In contrast, CYP2B37 showed the highest catalytic efficiency with 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), followed by 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC). CYP2B35 had no dealkylation activity with 7-MFC or 7-EFC. Furthermore, the new CYP2B-4-CPI-bound structures were used as templates for docking the 7-substituted coumarin derivatives, which revealed orientations consistent with the functional studies. In addition, the observation of multiple -Cl and -NH-π interactions of 4-CPI with the aromatic side chains in the CYP2B35 and CYP2B37 structures provides insight into the influence of such functional groups on CYP2B ligand binding affinity and specificity. To conclude, structural, computational, and functional analysis revealed striking differences between the active sites of CYP2B35 and CYP2B37 that will aid in the elucidation of new structure-activity relationships.
Asunto(s)
Cumarinas/química , Citocromo P-450 CYP2B1/química , Imidazoles/química , Modelos Moleculares , Xenobióticos/química , Animales , Sitios de Unión/fisiología , Cumarinas/metabolismo , Cristalografía por Rayos X , Citocromo P-450 CYP2B1/metabolismo , Imidazoles/metabolismo , Estructura Secundaria de Proteína , Ratas , Relación Estructura-Actividad , Xenobióticos/metabolismoRESUMEN
P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.
Asunto(s)
Inhibidores del Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/química , Simulación de Dinámica Molecular , Compuestos Orgánicos/química , Alcaloides de Triptamina Secologanina/química , Dominio Catalítico , Cristalografía por Rayos X , Citocromo P-450 CYP2D6/genética , Humanos , Enlace de HidrógenoRESUMEN
X-ray crystal structures of complexes of cytochromes CYP2B6 and CYP2A6 with the monoterpene sabinene revealed two distinct binding modes in the active sites. In CYP2B6, sabinene positioned itself with the putative oxidation site located closer to the heme iron. In contrast, sabinene was found in an alternate conformation in the more compact CYP2A6, where the larger hydrophobic side chains resulted in a significantly reduced active-site cavity. Furthermore, results from isothermal titration calorimetry indicated a much more substantial contribution of favorable enthalpy to sabinene binding to CYP2B6 as opposed to CYP2A6, consistent with the previous observations with (+)-α-pinene. Structural analysis of CYP2B6 complexes with sabinene and the structurally similar (3)-carene and comparison with previously solved structures revealed how the movement of the F206 side chain influences the volume of the binding pocket. In addition, retrospective analysis of prior structures revealed that ligands containing -Cl and -NH functional groups adopted a distinct orientation in the CYP2B active site compared with other ligands. This binding mode may reflect the formation of Cl-π or NH-π bonds with aromatic rings in the active site, which serve as important contributors to protein-ligand binding affinity and specificity. Overall, the findings from multiple techniques illustrate how drugs metabolizing CYP2B6 and CYP2A6 handle a common hydrocarbon found in the environment. The study also provides insight into the role of specific functional groups of the ligand that may influence the binding to CYP2B6.
Asunto(s)
Citocromo P-450 CYP2A6/química , Citocromo P-450 CYP2B6/química , Monoterpenos/química , Compuestos Orgánicos Volátiles/química , Monoterpenos Bicíclicos , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , TermodinámicaRESUMEN
Knowing how the protein environment modulates ligand pathways and redox centers in the respiratory heme-copper oxidases is fundamental for understanding the relationship between the structure and function of these enzymes. In this study, we investigated the reactions of O2 and NO with the fully reduced G232V mutant of ba3 cytochrome c oxidase from Thermus thermophilus (Tt ba3) in which a conserved glycine residue in the O2 channel of the enzyme was replaced with a bulkier valine residue. Previous studies of the homologous mutant of Rhodobacter sphaeroides aa3 cytochrome c oxidase suggested that the valine completely blocked the access of O2 to the active site [Salomonsson, L., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 11617-11621]. Using photolabile O2 and NO carriers, we find by using time-resolved optical absorption spectroscopy that the rates of O2 and NO binding are not significantly affected in the Tt ba3 G232V mutant. Classical molecular dynamics simulations of diffusion of O2 to the active site in the wild-type enzyme and G232V mutant show that the insertion of the larger valine residue in place of the glycine appears to open up other O2 and NO exit/entrance pathways that allow these ligands unhindered access to the active site, thus compensating for the larger valine residue.
Asunto(s)
Proteínas Bacterianas/química , Grupo Citocromo b/química , Complejo IV de Transporte de Electrones/química , Glicina/química , Thermus thermophilus/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Grupo Citocromo b/genética , Complejo IV de Transporte de Electrones/genética , Ligandos , Simulación de Dinámica Molecular , Mutación , Óxido Nítrico/química , Oxidación-Reducción , Oxígeno/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Valina/químicaRESUMEN
X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance.
Asunto(s)
Membrana Celular/enzimología , Sistema Enzimático del Citocromo P-450/química , Proteínas de la Membrana/química , Animales , Carcinógenos/metabolismo , Dominio Catalítico , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Inactivación Metabólica , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Modelos Moleculares , Estructura Secundaria de Proteína , Transporte de ProteínasRESUMEN
Cytochrome P450 46A1 (CYP46A1) is the cholesterol 24-hydroxylase initiating the major pathways of cholesterol removal from the brain, and bicalutamide (BIC) is a drug of choice for the treatment of progressive androgen-dependent prostate cancer. We evaluated the interactions of BIC with CYP46A1 by x-ray crystallography and by conducting solution and mutagenesis studies. Because BIC is administered to patients as a racemic mixture of the S and R isomers, we studied all three, racemic BIC as well as the S and R isomers. Co-crystallization of CYP46A1 with racemic BIC led to structure determinations at 2.1 Å resolution with the drug complexes exhibiting novel properties. Both BIC isomers bind to the CYP46A1 active site in very similar single orientation and adopt an energetically unfavorable folded conformation. This folded BIC conformation is correlated with drug-induced localized shifts of amino acid side chains in CYP46A1 and unusual interactions in the CYP46A1-BIC complex. One of these interactions involves a water molecule that is coordinated to the P450 heme iron and also hydrogen-bonded to the BIC nitrile. Due to polarization of the water in this environment, the heme elicits previously unreported types of P450 spectral responses. We also observed that access to the P450 active site was affected by differential recognition of S versus R isomers at the CYP46A1 surface arising from BIC conformational polymorphism.
Asunto(s)
Anilidas/farmacología , Antineoplásicos/farmacología , Nitrilos/farmacología , Esteroide Hidroxilasas/metabolismo , Compuestos de Tosilo/farmacología , Animales , Encéfalo/metabolismo , Dominio Catalítico , Bovinos , Colesterol/metabolismo , Colesterol 24-Hidroxilasa , Hemo/química , Humanos , Hierro/química , Cinética , Microsomas/metabolismo , Modelos Químicos , Conformación Molecular , Mutagénesis Sitio-Dirigida , Mutación , Unión Proteica , Espectrofotometría/métodos , Estereoisomerismo , Esteroide Hidroxilasas/química , Agua/químicaRESUMEN
The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and k(inact) are 0.2 and 0.25 min(-1), respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and an S(50) of 4.5 µM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by electrospray ionization liquid chromatography and mass spectrometry, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206, leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low-affinity site being the catalytic site and the high-affinity site on the protein periphery. Computer-aided docking and molecular dynamics simulations with one or two ligands bound revealed that the high-affinity site is situated at the entrance of a substrate access channel surrounded by the F' helix, ß1-ß2 loop, and ß4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/metabolismo , Fenantrenos/farmacología , Regulación Alostérica/efectos de los fármacos , Animales , Hidrocarburo de Aril Hidroxilasas/química , Dominio Catalítico , Cristalografía por Rayos X , Familia 2 del Citocromo P450 , Cinética , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica/efectos de los fármacos , ConejosRESUMEN
Knowledge of the structure and dynamics of the ligand channel(s) in heme-copper oxidases is critical for understanding how the protein environment modulates the functions of these enzymes. Using photolabile NO and O(2) carriers, we recently found that NO and O(2) binding in Thermus thermophilus (Tt) ba(3) is ~10 times faster than in the bovine enzyme, indicating that inherent structural differences affect ligand access in these enzymes. Using X-ray crystallography, time-resolved optical absorption measurements, and theoretical calculations, we investigated ligand access in wild-type Tt ba(3) and the mutants, Y133W, T231F, and Y133W/T231F, in which tyrosine and threonine in the O(2) channel of Tt ba(3) are replaced by the corresponding bulkier tryptophan and phenylalanine, respectively, present in the aa(3) enzymes. NO binding in Y133W and Y133W/T231F was found to be 5 times slower than in wild-type ba(3) and the T231F mutant. The results show that the Tt ba(3) Y133W mutation and the bovine W126 residue physically impede NO access to the binuclear center. In the bovine enzyme, there is a hydrophobic "way station", which may further slow ligand access to the active site. Classical simulations of diffusion of Xe to the active sites in ba(3) and bovine aa(3) show conformational freedom of the bovine F238 and the F231 side chain of the Tt ba(3) Y133W/T231F mutant, with both residues rotating out of the ligand channel, resulting in no effect on ligand access in either enzyme.
Asunto(s)
Proteínas Bacterianas/química , Complejo IV de Transporte de Electrones/química , Miocardio/enzimología , Óxido Nítrico/química , Oxígeno/química , Thermus thermophilus/enzimología , Secuencias de Aminoácidos , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/genética , Dominio Catalítico , Bovinos , Cobre/química , Complejo IV de Transporte de Electrones/genética , Cinética , Ligandos , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Xenón/químicaRESUMEN
Although there are currently three generations of antifungal azoles on the market, even the third-generation agents show undesirable interactions with human cytochrome P450 (P450) enzymes. CYP46A1 is a cholesterol-metabolizing P450 in the brain that tightly binds a number of structurally distinct azoles. Previously, we determined the crystal structures of CYP46A1 in complex with voriconazole and clotrimazole, and in the present work we cocrystallized the P450 with posaconazole at 2.5 Å resolution. This long antifungal drug coordinates the P450 heme iron with the nitrogen atom of its terminal azole ring and adopts a linear configuration occupying the whole length of the substrate access channel and extending beyond the protein surface. Numerous drug-protein interactions determine the submicromolar Kd of posaconazole for CYP46A1. We compared the crystal structure of posaconazole-bound CYP46A1 with those of the P450 in complex with other drugs, including the antifungal voriconazole and clotrimazole. We also analyzed the accommodation of posaconazole in the active site of the target enzymes, CYPs 51, from several pathogenic species. These and the solution studies with different marketed azoles, collectively, allowed us to identify the determinants of tight azole binding to CYP46A1 and generate an overall picture of azole binding to this important P450. The data obtained suggest that development of CYP51-specific antifungal agents will continue to be a challenge. Therefore, structural understanding of the azole binding not only to CYPs 51 from the pathogenic species but also to different human P450s is required to deal efficiently with this challenge.
Asunto(s)
Antifúngicos/química , Azoles/química , Colesterol/metabolismo , Esteroide Hidroxilasas/química , Esteroide Hidroxilasas/metabolismo , Antifúngicos/farmacología , Azoles/farmacología , Sitios de Unión , Colesterol 24-Hidroxilasa , Clotrimazol/química , Clotrimazol/farmacología , Cristalografía por Rayos X/métodos , Sistema Enzimático del Citocromo P-450/metabolismo , Humanos , Unión Proteica , Pirimidinas/química , Pirimidinas/farmacología , Esterol 14-Desmetilasa/metabolismo , Triazoles/química , Triazoles/farmacología , VoriconazolRESUMEN
To identify the structural features underlying the distinct substrate and inhibitor profiles of P450 2C19 relative to the closely related human enzymes, P450s 2C8 and 2C9, the atomic structure (Protein Data Bank code 4GQS) of cytochrome P450 2C19 complexed with the inhibitor (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)methanone (Protein Data Bank chemical component 0XV) was determined to 2.87 Å resolution by x-ray crystallography. The conformation of the peptide backbone of P450 2C19 is most similar to that of P450 2C8, but the substrate-binding cavity of P450 2C8 is much larger than that of P450 2C19 due to differences in the amino acid residues that form the substrate-binding cavities of the two enzymes. In contrast, the substrate-binding cavity of P450 2C19 is much more similar in size to that of the structure of the P450 2C9 flurbiprofen complex than to that of a modified P450 2C9 or that of P450 2C8. The cavities of the P450 2C19 0XV complex and the P450 2C9 flurbiprofen complex differ, however, because the helix B-C loops of the two enzymes are dissimilar. These conformational differences reflect the effects of adjacent structural elements that interact with the B-C loops and that differ between the two enzymes. The availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding to this enzyme.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/química , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Citocromo P-450 CYP2C19 , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2C9 , Inhibidores Enzimáticos/química , Humanos , Modelos Moleculares , Conformación ProteicaRESUMEN
Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 Å resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for ß sheet 1, helices A, F, F', G", G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F' that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6.
Asunto(s)
Citocromo P-450 CYP2D6/química , Citocromo P-450 CYP2D6/metabolismo , Inhibidores Enzimáticos/metabolismo , Compuestos Orgánicos/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Inhibidores del Citocromo P-450 CYP2D6 , Humanos , Ligandos , Modelos Moleculares , Unión ProteicaRESUMEN
The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a(3) moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba(3)-oxidase from Thermus thermophilus, determined at ~2.8-3.2Å resolution, reveal a Fe-C distance of ~2.0Å, a Cu-O distance of 2.4Å and a Fe-C-O angle of ~126°. Upon photodissociation at 100K, X-ray structures indicate loss of Fe(a3)-CO and appearance of Cu(B)-CO having a Cu-C distance of ~1.9Å and an O-Fe distance of ~2.3Å. Absolute FTIR spectra recorded from single crystals of reduced ba(3)-CO that had not been exposed to X-ray radiation, showed several peaks around 1975cm(-1); after photolysis at 100K, the absolute FTIR spectra also showed a significant peak at 2050cm(-1). Analysis of the 'light' minus 'dark' difference spectra showed four very sharp CO stretching bands at 1970cm(-1), 1977cm(-1), 1981cm(-1), and 1985cm(-1), previously assigned to the Fe(a3)-CO complex, and a significantly broader CO stretching band centered at ~2050cm(-1), previously assigned to the CO stretching frequency of Cu(B) bound CO. As expected for light propagating along the tetragonal axis of the P4(3)2(1)2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba(3) crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO(2) at 2337cm(-1) and one from traces of CO at 2133cm(-1); while bands associated with CO bound to either Fe(a3) or to Cu(B) in "light" minus "dark" FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2Å and FTIR spectra support the long-held position that photolysis of Fe(a3)-CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu(B) atom; Fe(a3) and Cu(B) ligation, at the resolutions reported here, are otherwise unaltered.
Asunto(s)
Proteínas Bacterianas/química , Monóxido de Carbono/química , Grupo Citocromo b/química , Complejo IV de Transporte de Electrones/química , Hierro/química , Thermus thermophilus/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Monóxido de Carbono/metabolismo , Dominio Catalítico , Cobre/química , Cobre/metabolismo , Cristalografía por Rayos X , Grupo Citocromo b/genética , Grupo Citocromo b/metabolismo , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Compuestos Ferrosos/química , Compuestos Ferrosos/metabolismo , Hemo/química , Hemo/metabolismo , Hierro/metabolismo , Modelos Moleculares , Mutación , Fotólisis , Conformación Proteica/efectos de la radiación , Espectroscopía Infrarroja por Transformada de Fourier , Thermus thermophilus/genética , Rayos XRESUMEN
AutoDrug is software based upon the scientific workflow paradigm that integrates the Stanford Synchrotron Radiation Lightsource macromolecular crystallography beamlines and third-party processing software to automate the crystallography steps of the fragment-based drug-discovery process. AutoDrug screens a cassette of fragment-soaked crystals, selects crystals for data collection based on screening results and user-specified criteria and determines optimal data-collection strategies. It then collects and processes diffraction data, performs molecular replacement using provided models and detects electron density that is likely to arise from bound fragments. All processes are fully automated, i.e. are performed without user interaction or supervision. Samples can be screened in groups corresponding to particular proteins, crystal forms and/or soaking conditions. A single AutoDrug run is only limited by the capacity of the sample-storage dewar at the beamline: currently 288 samples. AutoDrug was developed in conjunction with RestFlow, a new scientific workflow-automation framework. RestFlow simplifies the design of AutoDrug by managing the flow of data and the organization of results and by orchestrating the execution of computational pipeline steps. It also simplifies the execution and interaction of third-party programs and the beamline-control system. Modeling AutoDrug as a scientific workflow enables multiple variants that meet the requirements of different user groups to be developed and supported. A workflow tailored to mimic the crystallography stages comprising the drug-discovery pipeline of CoCrystal Discovery Inc. has been deployed and successfully demonstrated. This workflow was run once on the same 96 samples that the group had examined manually and the workflow cycled successfully through all of the samples, collected data from the same samples that were selected manually and located the same peaks of unmodeled density in the resulting difference Fourier maps.
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Cristalografía por Rayos X/métodos , Descubrimiento de Drogas/métodos , Programas Informáticos , Automatización , Cristalografía por Rayos X/instrumentación , Modelos Moleculares , Sincrotrones , Interfaz Usuario-Computador , Flujo de TrabajoRESUMEN
Despite recent advances in atomic-level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-α-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-α-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-α-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-α-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme-ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility.
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Hidrocarburo de Aril Hidroxilasas/química , Monoterpenos/química , Termodinámica , Hidrocarburo de Aril Hidroxilasas/metabolismo , Monoterpenos Bicíclicos , Sitios de Unión , Citocromo P-450 CYP2B6 , Humanos , Modelos Moleculares , Estructura Molecular , Monoterpenos/metabolismoRESUMEN
An X-ray crystal structure of CYP2B4 in complex with the drug paroxetine [(3S,4R)-3-[(2H-1,3-benzodioxol-5-yloxy)methyl]-4-(4-fluorophenyl)piperidine] was solved at 2.14 Å resolution. The structure revealed a conformation intermediate to that of the recently solved complex with amlodipine and that of the more compact complex with 4-(4-chlorophenyl)imidazole in terms of the placement of the F-G cassette. Moreover, comparison of the new structure with 15 previously solved structures of CYP2B4 revealed some new insights into the determinants of active-site size and shape. The 2B4-paroxetine structure is nearly superimposable on a previously solved closed structure in a ligand-free state. Despite the overall conformational similarity among multiple closed structures, the active-site cavity volume of the paroxetine complex is enlarged. Further analysis of the accessible space and binding pocket near the heme reveals a new subchamber that resulted from the movement of secondary structural elements and rearrangements of active-site side chains. Overall, the results from the comparison of all 16 structures of CYP2B4 demonstrate a cluster of protein conformations that were observed in the presence or absence of various ligands.
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Antidepresivos de Segunda Generación/química , Hidrocarburo de Aril Hidroxilasas/química , Inhibidores Enzimáticos/química , Modelos Moleculares , Paroxetina/química , Sustitución de Aminoácidos , Animales , Antidepresivos de Segunda Generación/metabolismo , Antidepresivos de Segunda Generación/farmacología , Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Hidrocarburo de Aril Hidroxilasas/genética , Hidrocarburo de Aril Hidroxilasas/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , Bases de Datos de Proteínas , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Ligandos , Conformación Molecular/efectos de los fármacos , Proteínas Mutantes/antagonistas & inhibidores , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Paroxetina/metabolismo , Paroxetina/farmacología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína/efectos de los fármacos , Conejos , Ratas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/química , Inhibidores Selectivos de la Recaptación de Serotonina/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacologíaRESUMEN
We use a form of "freeze-trap, kinetic crystallography" to explore the migration of Xe atoms away from the dinuclear heme a(3)/Cu(B) center in Thermus thermophilus cytochrome ba(3) oxidase. This enzyme is a member of the heme-copper oxidase superfamily and is thus crucial for dioxygen-dependent life. The mechanisms involved in the migration of oxygen, water, electrons, and protons into and/or out of the specialized channels of the heme-copper oxidases are generally not well understood. Pressurization of crystals with Xe gas previously revealed a O(2) diffusion channel in cytochrome ba(3) oxidase that is continuous, Y-shaped, 18-20 Å in length and comprised of hydrophobic residues, connecting the protein surface within the bilayer to the a(3)-Cu(B) center in the active site. To understand movement of gas molecules within the O(2) channel, we performed crystallographic analysis of 19 Xe laden crystals freeze-trapped in liquid nitrogen at selected times between 0 and 480 s while undergoing outgassing at room temperature. Variation in Xe crystallographic occupancy at five discrete sites as a function of time leads to a kinetic model revealing relative degrees of mobility of Xe atoms within the channel. Xe egress occurs primarily through the channel formed by the Xe1 â Xe5 â Xe3 â Xe4 sites, suggesting that ingress of O(2) is likely to occur by the reverse of this process. The channel itself appears not to undergo significant structural changes during Xe migration, thereby indicating a passive role in this important physiological function.
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Grupo Citocromo b/química , Grupo Citocromo b/metabolismo , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Oxígeno/metabolismo , Xenón/química , Transporte Biológico , Grupo Citocromo b/genética , Complejo IV de Transporte de Electrones/genética , Congelación , Cinética , Modelos Moleculares , Conformación Proteica , Estructura Terciaria de Proteína , Temperatura , Thermus thermophilus/enzimologíaRESUMEN
Structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine have been determined by X-ray crystallography. The presence of two drug molecules suggests clear substrate access channels in each P450. According to a previously established nomenclature, amlodipine molecules were trapped in access pathway 2f in P450 2B6 and in pathway 2a or 2f in P450 2B4. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described solvent channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B', F, F', and G' and part of helix G is substantially more open in the amlodipine complexes than in the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F', and B' and the ß4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states not observed with previous P450 2B structures and provide clear evidence of the substrate access channels in two drug-metabolizing P450s. In addition, the structures exhibit the versatility that can be exploited via in silico studies with other P450 2B6 ligands as large as raloxifene and itraconazole.