Effect of Conformational Dynamics on Substrate Recognition and Specificity as Probed by the Introduction of a de Novo Disulfide Bond into Cytochrome P450 2B1

The conformational dynamics of cytochrome P450 2B1 (CYP2B1) were investigated through the introduction of a disulfide bond to link the I- and K-helices by generation of a double Cys variant, Y309C/S360C. The consequences of the disulfide bonding were examined both experimentally and in silico by molecular dynamics simulations. Under high hydrostatic pressures, the partial inactivation volume for the Y309C/S360C variant was determined to be −21 cm³mol⁻¹, which is more than twice as much as those of the wild type (WT) and single Cys variants (Y309C, S360C). This result indicates that the engineered disulfide bond has substantially reduced the protein plasticity of the Y309C/S360C variant. Under steady-state turnover conditions, the S360C variant catalyzed the N-demethylation of benzphetamine and O-deethylation of 7-ethoxy-trifluoromethylcoumarin as the WT did, whereas the Y309C variant retained only 39% of the N-demethylation activity and 66% of the O-deethylation activity compared with the WT. Interestingly, the Y309C/S360C variant restored the N-demethylation activity to the same level as that of the WT but decreased the O-deethylation activity to only 19% of the WT. Furthermore, the Y309C/S360C variant showed increased substrate specificity for testosterone over androstenedione. Molecular dynamics simulations revealed that the engineered disulfide bond altered substrate access channels. Taken together, these results suggest that protein dynamics play an important role in regulating substrate entry and recognition.Liver microsomal cytochromes P450 (CYP or P450)² metabolize a large number of clinically used drugs that have diverse steric and functional moieties. Despite low sequence homology among CYPs from different families, all P450s invariably contain a heme cofactor that is coordinated to a thiolate and catalyze the oxidative metabolism, mostly through hydroxylation, of substrates. However, production of reactive intermediates by P450s is often associated with drug toxicity and carcinogenesis, and inhibition or induction of a specific P450 isoform may lead to adverse drug-drug interactions (1). From a clinical and pharmacological perspective, it is important to understand the structure, function, and dynamics of P450s.Structural studies of P450s by x-ray crystallography in the past decade have provided us with a wealth of information regarding the structural organization, critical active site residues, and proton delivery pathways of P450s (2–4). In particular, these structural analyses have consistently shown that certain regions of the P450 structures such as the F/G and B/B′-C loops are extremely flexible and can undergo large conformational changes to accommodate substrates of various sizes, although the overall folding pattern of all P450s is conserved. For instance, an open conformation was observed in the ligand-free CYP2B4 crystal structure, whereas a closed conformation was reported for the CPI-bound CYP2B4 (3, 5). The open-to-closed conformational change involves large motions of the F- and G-helices and the F/G and B/B′-C loops. Based on comparisons of the crystal structures of CYP2B4 bound with inhibitors of different sizes, Zhao et al. (6) identified five plastic regions in P450s, including the B/B′-C loop (PR2) and F/G loop (PR4). Binding of ketoconazole or erythromycin to CYP3A4 led to a large increase in the active site volume (>80% increase) because of conformational changes primarily in the PR4, but interestingly the F- and G-helices moved in the opposite direction (7). These authors proposed that the extreme flexibility of CYP3A4 accounts for its promiscuity, as CYP3A4 metabolizes nearly ∼50% of all clinically used drugs. The complexity of the conformational flexibility and dynamics are also revealed in an MD simulation study of CYP3A4, 2C9 and 2A6 (8). Importantly, this molecular dynamics (MD) simulation study shows that the three-dimensional structure of P450s is more flexible in solution than was observed in the crystal structure.Despite intensive studies of the crystal structures of microsomal P450s, insights into the conformational dynamics of P450s in solution, particularly in relation to their functional importance, are lacking. A laser flash photolysis study of CO rebinding to CYP2E1 in solution revealed that the binding of substrates such as ethanol, pyrazole, and acetaminophen restricts the conformational flexibility of CYP2E1, as the kinetics for the rebinding of CO to ligand-bound CYP2E1 are significantly slower than those for the ligand-free CYP2E1 (9). A solution thermodynamics study of CYP2B4 supports the notion that CYP2B4 is remarkably flexible, as the entropy substantially decreases upon inhibitor binding resulting from reduction of the hydrophobic surface (10). In this study, a de novo disulfide bond is engineered into CYP2B1 and the consequences resulting from the disulfide bonding are examined both experimentally and in silico using MD simulations. To discern the effect of the de novo disulfide bond apart from the Cys mutagenesis, both the single and double Cys variants were characterized in detail. To our knowledge, this is the first report that investigates the consequences of limiting conformational dynamics in a P450 by incorporating a disulfide bond. Our results demonstrate that protein dynamics play an important role in regulating substrate entry/product egress channels and substrate recognition and provide insights that will be valuable for rational drug design and protein engineering.