Molecular modeling of mammalian cytochrome P450s (original) (raw)
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Substrate recognition by two different P450s: Evidence for conserved roles in a common fold OPEN
Cytochrome P450 monooxygenases CYP101A1 and MycG catalyze regio-and stereospecific oxidations of their respective substrates, d-camphor and mycinamicin IV. Despite the low sequence homology between the two enzymes (29% identity) and differences in size and hydrophobicity of their substrates, the conformational changes that occur upon substrate binding in both enzymes as determined by solution NMR methods show some striking similarities. Many of the same secondary structural features in both enzymes are perturbed, suggesting the existence of a common mechanism for substrate binding and recognition in the P450 superfamily. Cytochrome P450 monooxygenases typically activate molecular oxygen, enabling O 2 to react with otherwise unactivated C-H and CC bonds in their substrates. These oxidations can be highly regio-and stereoselective, and as such, P450s are often found in the biosynthetic pathways leading to complex bioactive molecules (steroid hormones, antibiotics, toxins) or in the breakdown of compounds that can be used as sources of carbon and reducing equivalents. The cytochrome P450 superfamily is vast, with nearly 500,000 entries in GenBank ascribed to P450s as of early 2017, and members of the superfamily having been characterized for all kingdoms and nearly all phyla of life. Although many of these enzymes likely perform identical functions in different organisms, they nevertheless represent a vast array of potential substrate-product combinations, with substrates ranging in size from monoterpenes to macrocyclic and polycyclic compounds that exhibit a wide range of functionality and hydrophobicity. It is remarkable that despite this diversity, the P450 superfamily exhibits a unique and highly conserved fold, with identical topology and (usually) conserved secondary structural features identified in all of the P450 structures determined to date 1. Clearly, the P450 fold was arrived upon early in evolution as a safe platform for an inherently dangerous reaction, the activation of molecular oxygen 2. This dichotomy presents some fascinating questions: What structural or dynamic features must be conserved for the enzyme to safely activate O 2 ? What can be modified in order to fill a particular metabolic niche? Once a substrate is recognized and bound, how does the enzyme orient substrate appropriately for the observed chemistry? Given this combination of functional diversity and structural/mechanistic conservatism, the P450 superfamily provides an excellent platform for understanding the role of structural and sequence variability in a conserved enzyme function. We have found that nuclear magnetic resonance (NMR) can provide satisfying answers to some of the questions raised above. While X-ray crystallography is the most efficient means of obtaining structural information for P450s, relating the crystal structure to mechanism is not always straightforward. Substrate orientations may not rationalize the observed chemistry 3 , and it is not a given where on the reaction pathway the enzyme con-formation that crystallizes may lie, or even if it is mechanistically relevant. Solution NMR permits tight control of conditions (e.g., oxidation state, ligands/substrates, ionic strength) so that the relevance of structure and dynamics to a given point in the reaction pathway can be more readily assessed 1. Chemical shift perturbation and dynamics measurements provide insight into those structural features that are involved in substrate and effector recognition 4–8 , and residual dipolar couplings (RDCs) can be used to characterize enzyme conformations that are populated as a function of substrate or effector binding 9–13. We have previously used RDC-directed " soft annealing " molecular dynamics simulations to show that CYP101A1 (cytochrome P450 cam) undergoes significant conformational changes upon binding of substrate d-camphor 11,12 , and via site-directed mutagenesis coupled with activity assays, demonstrated that the observed changes are not artifacts of the methodology, but reliable
Life sciences, 1997
In recent years, homology modeling has become an important tool to study cytochrome P450 function, especially in conjunction with experimental approaches such as site-directed mutagenesis. Molecular models of mammalian P450s can be constructed based on crystal structures of four bacterial enzymes, P450cam, P450 BM-3, P450terp and P450eryF, using molecular replacement or consensus methods. In a model built by molecular replacement, the coordinates are copied from those of a given template protein, while consensus methods utilize more then one protein as a template and are based on distance geometry calculations. The models can be used to identify or confirm key residues, evaluate enzyme-substrate interactions and explain changes in protein stability and/or regio- and stereospecificity of substrate oxidation upon residue substitution by site-directed mutagenesis. P450 models have also been utilized to analyze binding of inhibitors or activators, as well as alterations in inhibition an...
Using a homology model of cytochrome P450 2D6 to predict substrate site of metabolism
Journal of Computer-Aided Molecular Design, 2010
CYP2D6 is an important enzyme that is involved in first pass metabolism and is responsible for metabolizing ~25% of currently marketed drugs. A homology model of CYP2D6 was built using X-ray structures of ligand-bound CYP2C5 complexes as templates. This homology model was used in docking studies to rationalize and predict the site of metabolism of known CYP2D6 substrates. While the homology model was generally found to be in good agreement with the recently solved apo (ligand-free) X-ray structure of CYP2D6, significant differences between the structures were observed in the B' and F-G helical region. These structural differences are similar to those observed between ligand-free and ligand-bound structures of other CYPs and suggest that these conformational changes result from induced-fit adaptations upon ligand binding. By docking to the homology model using Glide, it was possible to identify the correct site of metabolism for a set of 16 CYP2D6 substrates 85% of the time when the 5 top scoring poses were examined. On the other hand, docking to the apo CYP2D6 X-ray structure led to a loss in accuracy in predicting the sites of metabolism for many of the CYP2D6 substrates considered in this study. These results demonstrate the importance of describing substrate-induced conformational changes that occur upon binding. The best results were obtained using Glide SP with van der Waals scaling set to 0.8 for both the receptor and ligand atoms. A discussion of putative binding modes that explain the distribution of metabolic sites for substrates, as well as a relationship between the number of metabolic sites and substrate size, are also presented. In addition, analysis of these binding modes enabled us to rationalize the typical hydroxylation and O-demethylation reactions catalyzed by CYP2D6 as well as the less common N-dealkylation.
Journal of computer- …, 2000
Three-dimensional models of the cytochromes P450 IA2, P450 IID6 and P450 IIIA4 were built by means of comparative modeling using the X-ray crystallographic structures of P450 CAM, P450 BM-3, P450 TERP and P450 ERYF as templates. The three cytochromes were analyzed both in their intrinsic structural features and in their interaction properties with fifty specific and non-specific substrates. Substrate/enzyme complexes were obtained by means of both automated rigid and flexible body docking. The comparative analysis of the three cytochromes and the selected substrates, in their free and bound forms, allowed for the building of semi-quantitative models of substrate specificity based on both molecular and intermolecular interaction descriptors. The results of this study provide new insights into the molecular determinants of substrate specificity for the three different eukaryotic P450 isozymes and constitute a useful tool for predicting the specificity of new compounds.
Molecular modeling of cytochrome P450 3A4
Journal of computer-aided molecular design, 1997
The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s; P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6 beta-orientation can interact with a number of active s...
Biochemistry, 2003
The structure of rabbit microsomal cytochrome P450 2C5/3LVdH complexed with a substrate, 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide (DMZ), was determined by X-ray crystallography to 2.3 Å resolution. Substrate docking studies and electron density maps indicate that DMZ binds to the enzyme in two antiparallel orientations of the long axis of the substrate. One orientation places the principal site of hydroxylation, the 4-methyl group, 4.4 Å from the heme Fe, whereas the alternate conformation positions the second, infrequent site of hydroxylation at >5.9 Å from the heme Fe. Comparison of this structure to that obtained previously for the enzyme indicates that the protein closes around the substrate and prevents open access of water from bulk solvent to the heme Fe. This reflects a ∼1.5 Å movement of the F and G helices relative to helix I. The present structure provides a complete model for the protein from residues 27-488 and defines two new helices F′ and G′. The G′ helix is likely to contribute to interactions of the enzyme with membranes. The relatively large active site, as compared to the volume occupied by the substrate, and the flexibility of the enzyme are likely to underlie the capacity of drug-metabolizing enzymes to metabolize structurally diverse substrates of different sizes. † This investigation was supported by NIH Grants GM31001 (E.F.J.) and GM59229 (C.D.S.) and by CNRS and the French Minister of Research (D.M.). ‡ Structural coordinates have been deposited with the Protein Data Bank under accession code 1N6B.
Cytochrome P450 side-chain cleavage: Insights gained from homology modeling
Molecular and Cellular Endocrinology, 2007
Cytochrome P450 side-chain cleavage (CYP11A1) catalyzes the conversion of cholesterol to pregnenolone, the first step in steroidogenesis. The absence of a solved crystal structure has complicated deductions pertaining to the structure/function relationships of this key enzyme. Although a number of techniques have been employed to identify domains and specific amino acid residues important for catalytic activity, these methods have been unsuccessful in predicting three-dimensional orientations in space and thus the mechanism by which they exert their kinetic effect. This review aims to demonstrate the significant contribution homology modelling, when employed as a tool in combination with other standard biochemical techniques, has made towards our understanding of CYP11A1.
Modelling of P450 active site based on consensus 3D structures
P450 enzymes constitute a large superfamily of haem-thiolate proteins involved in the metabolism of numerous substrates such as drugs, carcinogens and sex hormones. Description of the structure of cytochrome P450 active sites is a key element to design better drugs. However, to date there are less than 150 known structures of P450 proteins. The generation of biologically meaningful 3D patterns or motifs from the simultaneous alignment of several P450 structures is a way of overcoming that lack of data. In order to address this problem, we investigated the simultaneous structural alignments of these proteins. Our new method is based on the comparison of sets of homologue proteins the 3D structures of which are known. Proteins are aligned according to the position of their haem groups. Then, from that multiple alignment, a consensus 3D template is produced providing information concerning atom and chemical group positions as well as cavity location. Experiments on human CYP17 show that these templates contain biologically significant patterns and highlight residues involved in catalytic reactions. Moreover, our 3D templates prove consistent with models of CYP17 active sites generated independently. Therefore, these 3D templates could be exploited for drug design.
Elucidation of Distinct Ligand Binding Sites for Cytochrome P450 3A4 †
Biochemistry, 2000
Cytochrome P450 (P450) 3A4 is the most abundant human P450 enzyme and has broad selectivity for substrates. The enzyme can show marked catalytic regioselectivity and unusual patterns of homotropic and heterotropic cooperativity, for which several models have been proposed. Spectral titration studies indicated one binding site for the drug indinavir (M r 614), a known substrate and inhibitor. Several C-terminal aminated peptides, including the model morphiceptin (YPFP-NH 2 ), bind with spectral changes indicative of Fe-NH 2 bonding. The binding of the YPFP-NH 2 N-terminal amine and the influence of C-terminal modification on binding argue that the entire molecule (M r 521) fits within P450 3A4. YPFP-NH 2 was not oxidized by P450 3A4 but blocked binding of the substrates testosterone and midazolam, with K i values similar to the spectral binding constant (K s ) for YPFP-NH 2 . YPFP-NH 2 inhibited the oxidations of several typical P450 substrates with K i values 10-fold greater than the K s for binding YPFP-NH 2 and its K i for inhibiting substrate binding. The n values for cooperativity of these oxidations were not altered by YPFP-NH 2 . YPFP-NH 2 inhibited the oxidations of midazolam at two different positions (1′-and 4-) with 20-fold different K i values. The differences in the K i values for blocking the binding to ferric P450 3A4 and the oxidation of several substrates may be attributed to weaker binding of YPFP-NH 2 to ferrous P450 3A4 than to the ferric form. The ferrous protein can be considered a distinct form of the enzyme in binding and catalysis because many substrates (but not YPFP-NH 2 ) facilitate reduction of the ferric to ferrous enzyme. Our results with these peptides are considered in the context of several proposed models. A P450 3A4 model based on these peptide studies contains at least two and probably three distinct ligand sites, with testosterone and R-naphthoflavone occupying distinct sites. Midazolam appears to be able to bind to P450 3A4 in two modes, one corresponding to the testosterone binding mode and one postulated to reflect binding in a third site, distinct from both testosterone and R-naphthoflavone. The work with indinavir and YPFP-NH 2 also argues that room should be present in P450 3A4 to bind more than one smaller ligand in the "testosterone" site, although no direct evidence for such binding exists. Although this work with peptides provides evidence for the existence of multiple ligand binding sites, the results cannot be used to indicate their juxtaposition, which may vary through the catalytic cycle.