title: The use of isomeric testosterone dimers to explore allosteric effects in substrate binding to cytochrome P450 CYP3A4.
abstract: Cytochrome P450 CYP3A4 is the main drug-metabolizing enzyme in the human liver, being responsible for oxidation of 50% of all pharmaceuticals metabolized by human P450 enzymes. Possessing a large substrate binding pocket, it can simultaneously bind several substrate molecules and often exhibits a complex pattern of drug-drug interactions. In order to better understand structural and functional aspects of binding of multiple substrate molecules to CYP3A4 we used resonance Raman and UV-VIS spectroscopy to document the effects of binding of synthetic testosterone dimers of different configurations, cis-TST2 and trans-TST2. We directly demonstrate that the binding of two steroid molecules, which can assume multiple possible configurations inside the substrate binding pocket of monomeric CYP3A4, can lead to active site structural changes that affect functional properties. Using resonance Raman spectroscopy, we have documented perturbations in the ferric and Fe-CO states by these substrates, and compared these results with effects caused by binding of monomeric TST. While the binding of trans-TST2 yields results similar to those obtained with monomeric TST, the binding of cis-TST2 is much tighter and results in significantly more pronounced conformational changes of the porphyrin side chains and Fe-CO unit. In addition, binding of an additional monomeric TST molecule in the remote allosteric site significantly improves binding affinity and the overall spin shift for CYP3A4 with trans-TST2 dimer bound inside the substrate binding pocket. This result provides the first direct evidence for an allosteric effect of the peripheral binding site at the protein-membrane interface on the functional properties of CYP3A4.
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, United States. Electronic address: denisov@illinois.edu.
authors.forename: Ilia G
authors.initials: IG
authors.lastname: Denisov
authors.affiliation: Department of Chemistry, Marquette University, Milwaukee, WI 53233, United States. Electronic address: piotr.mak@marquette.edu.
authors.forename: Piotr J
authors.initials: PJ
authors.lastname: Mak
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, United States. Electronic address: grinkova@illinois.edu.
authors.forename: Yelena V
authors.initials: YV
authors.lastname: Grinkova
authors.affiliation: Département de chimie, biochimie et physique, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada. Electronic address: Dominic.Bastien@uqtr.ca.
authors.forename: Dominic
authors.initials: D
authors.lastname: Bastien
authors.affiliation: Département de chimie, biochimie et physique, Université du Québec à Trois-Rivières, Trois-Rivières, Québec G9A 5H7, Canada. Electronic address: Gervais.Berube@uqtr.ca.
authors.forename: Gervais
authors.initials: G
authors.lastname: Bérubé
authors.affiliation: Department of Biochemistry, University of Illinois, Urbana, IL 61801, United States; Department of Chemistry, University of Illinois, Urbana, IL 61801, United States. Electronic address: s-sligar@illinois.edu.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Sligar
authors.affiliation: Department of Chemistry, Marquette University, Milwaukee, WI 53233, United States. Electronic address: james.kincaid@marquette.edu.
authors.forename: James R
authors.initials: JR
authors.lastname: Kincaid
mesh_terms: D000495:Allosteric Site; D001665:Binding Sites; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D013059:Spectrum Analysis, Raman; D013739:Testosterone / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism
chemical_list: D013739:Testosterone; C020290:dimeric testosterone; D051544:Cytochrome P-450 CYP3A
keywords: Allosteric properties; CYP3A4; Cytochrome P450; Nanodiscs; Resonance Raman; Testosterone dimers