X‐ray Crystal Structure of the Cytochrome P450 2B4 Active Site Mutant F297A in Complex with Clopidogrel: Insights into Compensatory Rearrangements of the Binding Pocket

Abstract

Prior X‐ray crystal structures of rabbit cytochrome P450 2B4 and human P450 2B6 have revealed the pivotal role of side‐chain rearrangement of active site residues F206 and F297 in accommodating various inhibitors or substrates. To explore further the structural basis of ligand binding, the crystal structure of 2B4 F297A was solved in complex with the drug clopidogrel. The structure demonstrated the reorientation of ligand in the active site such that the acetoxy group is oriented toward the heme, whereas the thiophene moiety now extends to the void region of A297 on the I‐helix. Interestingly, movement of the I‐helix and several amino acid side chains within the active site was observed in apparent response to the altered binding orientation. Results of flexible docking using the 2B4 wild type or the F297A‐virtual mutant positioned either the thiophene or chlorophenyl group closer to heme. However, docking of clopidogrel using the F297A structure or a virtual mutant where the I‐helix was also repositioned, oriented clopidogrel preferentially with either the acetoxy or the chlorophenyl group closest to heme. The study provides insight into how the altered active site adapts to accommodate the substrate in a distinct orientation and has important implications for understanding the role of active site residues in functional differences among related P450 enzymes. Supported by NIH grant ES03619.