Xu, Y.; Zawlewski, A.; Pandey, A. V.

Human cytochrome P450 17A1 (CYP17A1) is the qualitative regulator of steroidogenesis, catalyzing both 17-hydroxylation and 17,20-lyase cleavage at a single active site, and is the target of the prostate cancer drug abiraterone. Crystal structures show an almost fully enclosed site and do not reveal how lipophilic steroids reach it from the endoplasmic reticulum membrane or how the more polar products leave. We therefore asked which routes connect the CYP17A1 active site to the protein surface, and which of them are required. Molecular dynamics of the CYP17A1 enzyme followed by CAVER tunnel clustering produced 99 transport pathways in 14 clusters, which grouped by the elements forming their mouths into four topologically distinct portals (P-I to P-IV). Five structure-guided variants of CYP17A1 were expressed in HEK293 cells and both hydroxylase and lyase reactions were measured. Occluding the dominant P-I/P-II bottleneck (A105L) left both reactions intact and slightly raised them (116.1 {+/-} 8.0% hydroxylase, P = 0.0043; 134.8 {+/-} 27.9% lyase, P = 0.028). A tryptophan in the F/G lumen (L242W) was tolerated (96.6{+/-} 6.9% and 89.9{+/-} 10.4%, neither significant), whereas an engineered I205C/R239C pair across the same route abolished catalysis (below 2%, P < 0.001). Both perturbations of the membrane-facing P-IV portal cut activity below 2% of wild type (P < 0.001), and CYP17A1 protein levels accounted for neither phenotype. These data support a dual-portal model of vectorial ligand transport in CYP17A1.