THU572 Structure, Organisation And Mechanistic Insight Of Cytochrome P450 17A1
Authors: Martin Lisandra L , O'Leary Paul , Arumugam Senthil , Bell Stephen , Rodgers Raymond J 
Published: October 2023

Disclosure: L.L. Martin: None. P. O'Leary: None. S. Arumugam: None. S. Bell: None. R.J. Rodgers: None.

Introduction: Cytochrome P45017A1 (CYP17) is a heme-containing, membrane bound enzyme situated at a key branch within steroidogenic pathways. CYP17 is a dual function enzyme that initially catalyses 17α-hydroxylation of pregnenolone and progesterone to 17α-hydroxypregnenolone and 17α-hydroxyprogesterone. CYP17 can further metabolize these steroids via 17,20-lyase reactions to produce androgen/estrogen precursors. Activity of CYP17 requires electrons delivered by cytochrome P450 oxido-reductase (CPR) and and accessory protein cytochrome b5 (cyt b5) for the 17,20-lyase reaction. Previously, we showed that (i) CYP17 could homodimerise and heterodimerise with CPR and cyt b5 in cells using FRET [1] and computer models [2] and (ii) protein-protein interaction of cyt b5 with CYP17 slowed the rate of electron transfer to CYP17 [3] by electrochemistry. The current study focuses on sub-cellular organisation of CYP17 and mechanistic insight gained via analysis of the structural and molecular interactions of CYP17 with its partner proteins associated with 17α-hydroxylase and 17,20-lyase reactions. Methods: Single molecule fluorescence microscopy (SMFM) was used to observe fluorescently tagged CYP17 in live HEK 293 cells. Molecular Dynamics (MD) simulations were done for CYP17 in membranes, in the presence and absence of substrates, while interacting with CPR and cyt b5. Results: Halo-tagged CYP17 was imaged in real-time within the endoplasmic reticulum, providing kinetic data and sub-cellular localisation dynamics of individual molecules.MD simulations, within a membrane environment, allowed the dynamics of full-length models of CYP17 to be simulated: (i) in the absence of substrate, (ii) with a hydroxylase substrate (pregnenolone), and (iii) with a lyase substrate (17α-OH pregnenolone). Intriguingly, the protein-protein interaction of cyt b5 decreased the movement and flexibility within the active site of CYP17, however, regions not necessarily obvious for haem-substrate rearrangement were also altered. Single molecule fluorescence revealed CYP17 molecules moved dynamically along the endoplasmic reticulum. Conclusions: MD analyses provided structural insight on the mechanism underlying the allosteric coupling between the cyt b5 to the CYP17 interface and specifically to the active site. Single molecule fluorescence tracking indicated that CYP17 actively moves dynamically in the endoplasmic reticulum.

1. Praporski, S., et al., Organization of Cyt P450 Enzymes Involved in Sex Steroid Synthesis. J. Biol. Chem., 2009. 284 33224-33232. 2. Holien, J.K., et al., A Homodimer Model Can Resolve the Conundrum as to How CPR and Cyt b5 Compete for the Same Binding Site on Cyt P450c17. Curr Protein Pept Sci, 2017. 18 515-521. 3. Simonov, A.N., et al., Mechanistic Scrutiny Identifies a Kinetic Role for Cyte b5 Regulation of Human Cyt P450c17. PLoS ONE, 2015. 10 e0141252.

Presentation Date: Thursday, June 15, 2023