Dual Ligand Complexes of Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4 with Amlodipine Reveal Substrate Access Channels into the Active Site

Abstract

Substrate access channels have been predicted previously for several mammalian P450 enzymes based mainly on molecular dynamics simulations. We now report X‐ray crystal structures of human P450 2B6 and rabbit P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine. The presence of the two drug molecules identifies clear access pathways in each P450. Based on the access channel nomenclature, 2f and 2a channels pointing towards the membrane correlate well with amlodipine access into the active site of P450 2B4. However, amlodipine appears to enter the active site of human P450 2B6 only via channel 2f. The results also indicate that key arginine residues located on the surface and at the entrance of the substrate access channels in each of the above P450s may play a crucial role in guiding substrate entry. In addition, secondary structure elements B′, F, F′, G′ and part of helix G were substantially retracted compared with the 4‐(4‐chlorophenyl)imidazole complexes of 2B4 and 2B6. Conformational variations were predominantly localized to the above elements that surround the substrate access channels as seen previously in the open P450cam structure. To our knowledge, the structures represent the first clear evidence from X‐ray crystallography of the substrate access channels into the active site in mammalian P450s. Supported by NIH grant ES03619.