title: Photo-initiated crosslinking extends mapping of the protein-protein interface to membrane-embedded portions of cytochromes P450 2B4 and b₅.
abstract: Protein-protein interactions play a central role in the regulation of many biochemical processes (e.g. the system participating in enzyme catalysis). Therefore, a deeper understanding of protein-protein interactions may contribute to the elucidation of many biologically important mechanisms. For this purpose, it is necessary to establish the composition and stoichiometry of supramolecular complexes and to identify the crucial portions of the interacting molecules. This study is devoted to structure-functional relationships in the microsomal Mixed Function Oxidase (MFO) complex, which is responsible for biotransformation of many hydrophobic endogenous compounds and xenobiotics. In particular, the cytochrome b5 interaction with MFO terminal oxygenase cytochrome P-450 (P450) was studied. To create photolabile probes suitable for this purpose, we prepared cytochrome b5 which had a photolabile diazirine analog of methionine (pMet) incorporated into the protein sequence, employing recombinant expression in Escherichia coli. In addition to wild-type cytochrome b5, where three methionines (Met) are located at positions 96, 126, and 131, six mutants containing only one Met in the sequence were designed and expressed (see Table 1). In these mutants, a single Met was engineered into the catalytic domain (at positions 23, 41, or 46), into the linker between the protein domains (at position 96), or into the membrane region (at positions 126 or 131). These mutants should confirm or exclude these portions of cytochrome b5 which are involved in the interaction with P450. After UV irradiation, the pMet group(s) in the photolabile cytochrome b5 probe was(were) activated, producing covalent crosslinks with the interacting parts of P450 2B4 in the close vicinity. The covalent complexes were analyzed by the "bottom up" approach with high-accuracy mass spectrometry. The analysis provided an identification of the contacts in the supramolecular complex with low structural resolution. We found that all the above-mentioned cytochrome b5 Met residues can form intermolecular crosslinks and thus participate in the interaction. In addition, our results indicate the existence of at least two P450:cytochrome b5 complexes which differ in the orientation of individual proteins. The results demonstrate the advantages of the photo-initiated crosslinking technique which is able to map the protein-protein interfaces not only in the solvent exposed regions, but also in the membrane-embedded segments (compared to a typical crosslinking approach which generally only identifies crosslinks in solvent exposed regions).
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic; Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Tomáš
authors.initials: T
authors.lastname: Ječmen
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic; Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic.
authors.forename: Renata
authors.initials: R
authors.lastname: Ptáčková
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic.
authors.forename: Věra
authors.initials: V
authors.lastname: Černá
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Dračínská
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic.
authors.forename: Petr
authors.initials: P
authors.lastname: Hodek
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic.
authors.forename: Marie
authors.initials: M
authors.lastname: Stiborová
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic.
authors.forename: Jiří
authors.initials: J
authors.lastname: Hudeček
authors.affiliation: Department of Biochemistry, Faculty of Science, Charles University, Hlavova 2030, CZ-12843 Prague 2, Czech Republic; Institute of Microbiology v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-14220 Prague 4, Czech Republic. Electronic address: miroslav.sulc@natur.cuni.cz.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Šulc
mesh_terms: D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D010775:Photic Stimulation / Q000379:methods*; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000502:physiology; D011817:Rabbits
chemical_list: D003432:Cross-Linking Reagents; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)
keywords: CYP 2B4 and cytochrome b(5); Mass spectrometry; Mixed Function Oxidase system; Photo-initiated crosslinking; Protein–protein interaction; Trans-membrane segments