title: A large-scale allosteric transition in cytochrome P450 3A4 revealed by luminescence resonance energy transfer (LRET).
abstract: Effector-induced allosteric transitions in cytochrome P450 3A4 (CYP3A4) were investigated by luminescence resonance energy transfer (LRET) between two SH-reactive probes attached to various pairs of distantly located cysteine residues, namely the double-cysteine mutants CYP3A4(C64/C468), CYP3A4(C377/C468) and CYP3A4(C64/C121). Successive equimolar labeling of these proteins with the phosphorescent probe erythrosine iodoacetamide (donor) and the near-infrared fluorophore DY-731 maleimide (acceptor) allowed us to establish donor/acceptor pairs sensitive to conformational motions. The interactions of all three double-labeled mutants with the allosteric activators α-naphthoflavone and testosterone resulted in an increase in the distance between the probes. A similar effect was elicited by cholesterol. These changes in distance vary from 1.3 to 8.5 Å, depending on the position of the donor/acceptor pair and the nature of the effector. In contrast, the changes in the interprobe distance caused by such substrates as bromocriptine or 1-pyrenebutanol were only marginal. Our results provide a decisive support to the paradigm of allosteric modulation of CYP3A4 and indicate that the conformational transition caused by allosteric effectors increases the spatial separation between the beta-domain of the enzyme (bearing residues Cys64 and Cys377) and the alpha-domain, where Cys121 and Cys468 are located.
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America ; Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania, United States of America.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Sineva
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America ; Department of Chemistry and Biology, University of Waterloo, Waterloo, Ontario, Canada.
authors.forename: Jessica A O
authors.initials: JA
authors.lastname: Rumfeldt
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
authors.affiliation: Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California, United States of America.
authors.forename: Dmitri R
authors.initials: DR
authors.lastname: Davydov
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D001971:Bromocriptine / Q000494:pharmacology; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D008024:Ligands; D008163:Luminescent Measurements; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011721:Pyrenes / Q000494:pharmacology
chemical_list: C463480:1-pyrenebutanol; D008024:Ligands; D011721:Pyrenes; D001971:Bromocriptine; D006418:Heme; D051544:Cytochrome P-450 CYP3A