NMR-guided identification of CYP11A1–Adrenodoxin interactions that differentially govern cholesterol and vitamin D3 metabolism

Cytochromes P450 (CYPs) are heme-containing enzymes essential for a range of biochemical processes, including steroidogenesis and vitamin D metabolism. Among mitochondrial CYPs, CYP11A1 catalyzes both cholesterol side-chain cleavage, producing pregnenolone, and hydroxylation of vitamin D3, producing 20(OH)D3. Previous studies have shown that substrates can modulate CYP11A1 protein–protein interactions with the redox partner Adrenodoxin (Adx), but the structural basis of the substrate-specific modulation towards Adx is not known. In this study, we investigated whether there exist contact(s) between CYP11A1 and Adx that are differentially influenced by cholesterol and vitamin D3, and whether these substrate-specific contacts are important for CYP11A1 monooxygenation of vitamin D3 or side chain cleavage of cholesterol. Utilizing 2D NMR spectroscopy in combination with solubilization of substrates with hydroxypropyl-β-cyclodextrin, we were able to isolate M77 of Adx α helix-3 as a substrate-specific contact towards CYP11A1. Site-directed mutagenesis of Adx M77 into M77L and M77S and mutagenesis of the corresponding CYP11A1 contact (W418A) revealed differential effects towards cholesterol and vitamin D3 metabolism. These data suggest that CYP11A1 protein–protein interactions with Adx are uniquely driven by substrate specificity and shed light on potential substrate-sensitive recognition in other mitochondrial CYPs. These findings are further discussed in the context of a modeled interaction between CYP11A1 and the reduced (functional) form of Adx in which Adx M77–CYP11A1 W418 is the driving constraint. Moreover, this study describes an NMR-based protocol that is broadly applicable towards the investigation of other substrate-sensitive CYP–redox partner interactions.