Active-site differences between substrate-free and ritonavir-bound cytochrome P450 (CYP) 3A5 reveal plasticity differences between CYP3A5 and CYP3A4

Abstract

Cytochrome P450 (CYP) 3A4 is a major contributor to hepatic drug and xenobiotic metabolism in human adults. The related enzyme CYP3A5 is also expressed in adult liver and has broader age and tissue distributions. However, CYP3A5 expression is low in most Caucasians because of the prevalence of an allele that leads to an incorrectly spliced mRNA and premature termination of translation. When expressed, CYP3A5 expands metabolic capabilities and can augment CYP3A4-mediated drug metabolism, thereby reducing drug efficacy and potentially requiring dose adjustments. The extensive role of CYP3A4 in drug metabolism reflects in part the plasticity of the substrate-free enzyme to enlarge its active site and accommodate very large substrates. We have previously shown that the structure of the CYP3A5-ritonavir complex differs substantially from that of the CYP3A4-ritonavir complex. To better understand whether these differences are conserved in other CYP3A5 structures and how they relate to differential plasticity, we determined the X-ray crystallographic structure of the CYP3A5 substrate-free complex to 2.20 Å resolution. We observed that this structure exhibits a much larger active site than substrate-free CYP3A4 and displays an open substrate access channel. This reflected in part a lower trajectory of the helix F-F' connector in CYP3A4 and more extensive π-CH interactions between phenylalanine residues forming the roof of the active-site cavity than in CYP3A5. Comparison with the CYP3A5-ritonavir complex confirmed conserved CYP3A5 structural features and indicated differences in plasticity between CYP3A4 and CYP3A5 that favor alternative ritonavir conformations.

Keywords: CYP3A4; CYP3A5; crystal structure; cytochrome P450; drug metabolism; protein conformation; protein plasticity; protein structure.

Figure 1.

Conformational differences between apo 3A5 (yellow) and apo 3A4 (magenta; A and B), apo 3A5 and the 3A5 ritonavir complex (green; C and D), and apo 3A4 and the 3A4 ritonavir complex (cyan; E and F). Ritonavir is depicted as spheres. Heme is depicted as a stick model with an iron sphere. Heteroatom colors are nitrogen (blue), oxygen (red), iron (orange), and sulfur (yellow). Distances (dashed lines) are reported in angstroms.

Figure 2.

Active-site cavities are shown as semitransparent surfaces for apo 3A5 (A), 3A5 ritonavir complex (B), apo 3A4 (C), and 3A4 ritonavir complex (D) with adjacent amino acid side chains, the heme co-factor, and ritonavir are shown as stick models. Amino acid residues that differ between 3A4 and 3A5 are identified by bold labels. The viewpoint and scale are identical for each image.

Figure 3.

A view of the open entrance channel of the apo 3A5 active site with testosterone docked in the channel (A) and the closed entrance channel in apo 3A4 (B) that reflects the lower trajectory of F–F′ connector and interactions of Phe-107, Phe-215, and Phe-220 with Phe-213. The testosterone pose overlaps two glycerol molecules seen in the apo 3A5 structure (Fig. S1). The docking pose was identified by using Autodock Vina. The distance from testosterone (TES) to the heme iron is 16.7 Å. Amino acid residues that differ between 3A4 and 3A5 are identified by bold labels.

Figure 4.

Superposition of 3A4 (gray) with two molecules of ketoconazole (KLN) bound in the active site (PDB code 2V0M) with apo 3A5 (yellow) using ALIGN with defaults in PyMOL. Portions of the proteins are shown as cartoons displaying secondary and tertiary structure. The imidazole nitrogen of the lower ketoconazole molecule is ligated to the heme iron, and together with the neighboring dichlorophenyl group, they displace a portion of helix I outward. A second molecule of ketoconazole stacks above the first in anti-parallel orientation with dichlorophenyl moiety residing in the entrance channel. The conformation of the helix F–G regions of the two structures are overlapped to greater extent than seen for structures of the 3A5 and 3A4 ritonavir complexes, and the 3A4 active site is greatly enlarged by the presence of the two ketoconazole molecules.