title: Cross-linking mass spectrometry and mutagenesis confirm the functional importance of surface interactions between CYP3A4 and holo/apo cytochrome b(5).
abstract: Cytochrome b(5) (cyt b(5)) is one of the key components in the microsomal cytochrome P450 monooxygenase system. Consensus has not been reached about the underlying mechanism of cyt b(5) modulation of CYP catalysis. Both cyt b(5) and apo b(5) are reported to stimulate the activity of several P450 isoforms. In this study, the surface interactions of both holo and apo b(5) with CYP3A4 were investigated and compared for the first time. Chemical cross-linking coupled with mass spectrometric analysis was used to identify the potential electrostatic interactions between the protein surfaces. Subsequently, the models of interaction of holo/apo b(5) with CYP3A4 were built using the identified interacting sites as constraints. Both cyt b(5) and apo b(5) were predicted to bind to the same groove on CYP3A4 with close contacts to the B-B' loop of CYP3A4, a substrate recognition site. Mutagenesis studies further confirmed that the interacting sites on CYP3A4 (Lys96, Lys127, and Lys421) are functionally important. Mutation of these residues reduced or abolished cyt b(5) binding affinity. The critical role of Arg446 on CYP3A4 in binding to cyt b(5) and/or cytochrome P450 reductase was also discovered. The results indicated that electrostatic interactions on the interface of the two proteins are functionally important. The results indicate that apo b(5) can dock with CYP3A4 in a manner analogous to that of holo b(5), so electron transfer from cyt b(5) is not required for its effects.
authors.affiliation: Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195, USA.
authors.forename: Chunsheng
authors.initials: C
authors.lastname: Zhao
authors.forename: Qiuxia
authors.initials: Q
authors.lastname: Gao
authors.forename: Arthur G
authors.initials: AG
authors.lastname: Roberts
authors.forename: Scott A
authors.initials: SA
authors.lastname: Shaffer
authors.forename: Catalin E
authors.initials: CE
authors.lastname: Doneanu
authors.forename: Song
authors.initials: S
authors.lastname: Xue
authors.forename: David R
authors.initials: DR
authors.lastname: Goodlett
authors.forename: Sidney D
authors.initials: SD
authors.lastname: Nelson
authors.forename: William M
authors.initials: WM
authors.lastname: Atkins
mesh_terms: D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry; D019392:Cytochrome P-450 CYP2E1 / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015786:Cytochromes b5 / Q000737:chemistry* / Q000378:metabolism; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry; D013058:Mass Spectrometry; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D013250:Steroid Hydroxylases / Q000378:metabolism; D013739:Testosterone / Q000378:metabolism
chemical_list: D003432:Cross-Linking Reagents; D013739:Testosterone; D015786:Cytochromes b5; D013250:Steroid Hydroxylases; D019392:Cytochrome P-450 CYP2E1; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; D005022:Ethyldimethylaminopropyl Carbodiimide