Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Abstract

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.

Assessment of Arginine 97 and Lysine 72 as Determinants of Substrate Specificity in Cytochrome P450 2c9 (Cyp2c9)

“…D301 of CYP2D6 was believed to be responsible for interaction with substrate based on site-directed mutagenesis data using enzyme expressed in yeast (Ellis et al, 1995). However, D301 mutants appear to be unstable or poorly expressed in the bacterial system (Hanna et al, 2001), and recent data suggest that D301 may be involved in maintaining the structural integrity of the enzyme by stabilizing the B-C loop (Guengerich et al, 2002;Kirton et al, 2002;Paine et al, 2002). A role for D301 in direct interactions with some substrates cannot be excluded; however, an alteration in overall protein structure may indirectly perturb substrate interactions.…”

Why Is Quinidine an Inhibitor of Cytochrome P450 2D6?

“…The homology model of CYP2D6 was produced as described previously (14). In brief, the model was produced using the comparative modeling program Modeler (22) with five structural templates: cytochromes P450cam (23), P450terp (24), P450eryF (25), P450BM3 (26), and P4502C5 (27).…”

“…Modeling of Quinidine Binding to CYP2D6-To help rationalize the experimental data, computational docking studies were performed using our previously described model of wild-type CYP2D6 (14). 50 solutions for quinidine binding to oxygenated (compound I-like) and deoxygenated (type I-like) CYP2D6 were obtained, leading to solutions with good ChemScore values (Ϫ39.0 kJ⅐mol Ϫ1 ) (TABLE TWO), in keeping with the experimentally observed affinity (K d ϭ 0.4 M).…”

“…In the 3-carbon-tethered solutions, this was due to some unfavorable contacts, particularly with Phe 120 , Gln 216 , Thr 309 , and Phe 483 . However, Asp 301 is thought to contribute to the stabilization of the BЈ-C loop through a hydrogen bond with the backbone of Val 119 (14); and therefore, mutation of this residue to Gln could lead to a conformational change in the BЈ-C loop, thus impacting more globally on the nature of the active site.…”

“…14) suggest that two carboxylate groups (at Glu 216 and Asp 301 ) may play key roles in the recognition of substrates containing a basic nitrogen atom, and support for this has come from mutagenesis experiments (15)(16)(17). It has also been suggested that the aromatic residues Phe 120 , Phe 481 , and Phe 483 may have roles in substrate binding through -interactions with the planar hydrophobic regions common to many CYP2D6 substrates (10,14,18,19). Here, we describe studies of a series of mutants of these five residues aimed at investigating their role in quinidine binding and in determining whether quinidine is a substrate or an inhibitor of this important drug-metabolizing enzyme.…”