The F-G loop region of cytochrome P450scc (CYP11A1) interacts with the phospholipid membrane.
Cytochrome P450scc (CYP11A1) is a protein attached to the inner surface of the inner mitochondrial membrane that uses cholesterol from the membrane phase as its substrate for the first step in steroid hormone synthesis. We investigated the mechanism by which CYP11A1 interacts with the membrane. Hydrophobicity profiles of CYP11A1 and two other mitochondrial cytochromes P450, plus a model structure of CYP11A1 using CYP2C5 as template, suggest that CYP11A1 has a monotopic association with the membrane which may involve the A' helix and the F-G loop. Deletion of the A' helix reduced the proportion of expressed CYP11A1 associated with the bacterial membrane fraction, indicating a role for the A' helix in membrane binding. However, introduction of a cysteine residue in this helix at position 24 (L24C) and subsequent labelling with the fluorescent probe N'-(7-nitrobenz-2-oxal,3-diazol-4-yl)ethylenediamine (NBD) failed to show a membrane localisation. Cysteine mutagenesis and fluorescent labelling of other residues appearing on the distal surface of the CYP11A1 model revealed that V212C and L219C have enhanced fluorescence and a blue shift following association of the mutant CYP11A1 with phospholipid vesicles. This indicates that these residues, which are located in the F-G loop, become localised to a more hydrophobic environment following membrane binding. Analysis of the quenching of tryptophan residues in CYP11A1 by acrylamide indicates that at least one and probably two tryptophans are involved in membrane binding. We conclude that CYP11A1 has a monotopic association with the membrane that is mediated, at least in part, by the F-G loop region.
Headlam
Madeleine J
MJ
Department of Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia.
Wilce
Matthew C J
MC
Tuckey
Robert C
RC
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry* / Q000378:metabolism; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D046911:Macromolecular Substances; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010743:Phospholipids / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D008563:Membrane Lipids; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D002786:Cholesterol Side-Chain Cleavage Enzyme