Crystal Structure of CYP3A4 Complexed with Fluorol Identifies the Substrate Access Channel as a High-Affinity Ligand Binding Site

Abstract

Cytochrome P450 3A4 (CYP3A4) is a major human drug-metabolizing enzyme, notoriously known for its extreme substrate promiscuity, allosteric behavior, and implications in drug–drug interactions. Despite extensive investigations, the mechanism of ligand binding to CYP3A4 is not fully understood. We determined the crystal structure of CYP3A4 complexed with fluorol, a small fluorescent dye that can undergo hydroxylation. In the structure, fluorol associates to the substrate channel, well suited for the binding of planar polyaromatic molecules bearing polar groups, through which stabilizing H-bonds with the polar channel residues, such as Thr224 and Arg372, can be established. Mutagenesis, spectral, kinetic, and functional data confirmed the involvement but not strict requirement of Thr224 for the association of fluorol. Collectively, our data identify the substrate channel as a high-affinity ligand binding site and support the notion that hydrophobic ligands first dock to the nearby peripheral surface, before migrating to the channel and, subsequently, into the active site.

Unraveling the Ligand-Binding Sites of CYP3A4 by Molecular Dynamics Simulations with Solvent Probes

“…Obviously, sites A and 1 correspond to the active site above the heme group and the high-affinity site near the F′ helix, respectively. 16 These sites have been confirmed by X-ray crystallography. Site 2 is positioned in the triangular shaped area composed of the E, F, and G helices, which prompts a small pocket to accommodate various solvents.…”

“…13−15 In recent years, another novel binding site named the high-affinity site has been identified through crystal structure analysis. 16 The locations of these two sites are illustrated in Supporting Information Figure S1. Both biochemical and crystallographic studies support the presence of multiple ligand-binding sites in CYP3A4, implying that the binding sites may not be restricted to the two currently established sites.…”

“…The crystal structure of the progesterone-bound CYP3A4 complex reveals the position of the additional binding site other than the active site, which was named as the peripheral binding site . Extensive research has explored the relevance between the peripheral binding site and the allosteric effects subsequently. − In recent years, another novel binding site named the high-affinity site has been identified through crystal structure analysis . The locations of these two sites are illustrated in Supporting Information Figure S1.…”

Non-steroidal CYP17A1 Inhibitors: Discovery and Assessment

“…The structure showing that the fluorescent agent, fluorol, acts as an allosteric ligand and binds to a high-affinity binding site located in the substrate channel in CYP3A4 has recently been published. 51 It has been known for many years that the 17,20-lyase activity of CYP17A1 relative to the 17α-hydroxylase activity can be stimulated by phosphorylation or by binding of cyt b 5 . 52 Two mechanisms for the cyt b 5 interaction with CYP17A1 have been proposed.…”

“…Most of the studies have focused on CYP3A4, and it is proven without a doubt that this drug-metabolizing CYP contains an allosteric site. The structure showing that the fluorescent agent, fluorol, acts as an allosteric ligand and binds to a high-affinity binding site located in the substrate channel in CYP3A4 has recently been published …”