Abstract 1175 A Substrate Tug of War: How Cholesterol and Vitamin D3 Differentially Regulate CYP11A1 Structure and Redox Partner Interaction

Cytochrome P450 11A1 (CYP11A1) is a membrane associated mitochondrial enzyme that metabolizes both chemically distinct substrates cholesterol and vitamin D3, with less efficient metabolism towards the latter. The major pathway of CYP11A1 involves converting cholesterol to pregnenolone as the first and rate-limiting step in steroidogenesis. However, CYP11A1 vitamin D3 products have also been found in vivo, indicating an alternative pathway of vitamin D3 metabolism. CYP11A1 function relies on the delivery of two sequential electrons from Adrenodoxin (Adx). Recent studies suggest that both substrates and Adx play a regulatory role in CYP-mediated metabolism, leading us to hypothesize that vitamin D3 and cholesterol induce conformational change(s) of CYP11A1 that are relayed towards the proximal (Adx-binding) surface. In this study, our objective is to investigate how CYP11A1 substrates impact the CYP11A1 conformation and the CYP11A1-Adx redox partner interaction. We utilize 1D 19F-NMR as an in-solution technique that allows for the detection of multiple conformations of CYP11A1. 19F-NMR, which has not previously been applied to a mammalian CYP, allows us to side-step the size constraints and spectral crowding that result from conventional multi-dimensional NMR of a large enzyme (∼ 55 kDa). Preliminary 1D 19F-NMR spectra of CYP11A1 reveals substrate and Adx induced conformational changes that are consistent with structural regulation of the enzyme. We also combined 2D 1H-15N- Heteronuclear Single Quantum Coherence (HSQC) NMR with Biolayer interferometry (BLI) to investigate the nature of the protein-protein interactions with Adx. NMR titration of a pre-formed CYP11A1-Adx complex with β-cyclodextrin-incorporated cholesterol and vitamin D3 revealed a gradual focusing of NMR line-broadening effects towards α helix-3 of Adx and an overall decrease in the peak intensity remaining. Interestingly, only the cholesterol-CYP11A1 complex induced significant peak broadening to helix-1 of Adx, indicative of a substrate-specific contact. These findings are consistent with preliminary BLI data in which cholesterol increased the protein-protein interaction between CYP11A1 and Adx. In conclusion, these findings support substrate-specific modulation of the CYP11A-Adx protein-protein interaction as a regulatory feature of this enzyme. Understanding the structural basis of CYP11A1 regulation is critical in tissues where cholesterol (steroidogenic) and vitamin-D metabolic pathways intersect. This study was funded by NIH grant R35GM133375 (DFE).