Multiple molecular dynamics simulations of human p450 monooxygenase CYP2C9: The molecular basis of substrate binding and regioselectivity toward warfarin

Abstract To examine the molecular basis of activity and regioselectivity of the clinically important human microsomal cytochrome P450 (CYP) monooxygenase 2C9 toward its substrate warfarin, 22 molecular dynamics simulations (3–5 ns each) were performed in the presence and absence of warfarin. The resulting trajectories revealed a stable protein core and mobile surface elements. This mobility leads to the formation of two surface channels in the region between F‐G loop, B′ helix/B‐B′ loop, β 1 ‐sheet, and between helices F and I and the turn in the C‐terminal antiparallel β‐sheet in the presence of warfarin. Besides the nonproductive state of the CYP2C9 warfarin complex captured in the crystal structure, three additional states were observed. These states differ in the shape of the substrate binding cavity and the position of the warfarin molecule relative to heme. In one of these states, the 7‐ and 6‐positions of warfarin contact the heme with a marked geometrical preference for position 7 over position 6. This modeling result is consistent with experimentally determined regioselectivity (71 and 22% hydroxylation in positions 7 and 6, respectively). Access to the heme group is limited by the core amino acids Ala297, Leu362, Leu366, and Thr301, which therefore are expected to have a major impact on regioselectivity. In addition, modeling predicts that autoactivation of warfarin is sterically hindered. Our study demonstrates how the combination of mobile surface and rigid core leads to interesting properties: a broad substrate profile and simultaneously a high regioselectivity. Proteins 2006. © 2006 Wiley‐Liss, Inc.