The 26th International Symposium on Microsomes and Drug Oxidations

Human cytochrome P450 17A1 (CYP17A1) serves as the gatekeeper of steroidogenesis, partitioning precursors into glucocorticoid or androgen biosynthetic pathways and serving as a critical target for prostate cancer therapy. Despite the availability of static crystal structures, the dynamic pathways by which lipophilic substrates access the buried heme active site from the endoplasmic reticulum membrane, and how polar products exit to the cytosol, remain unresolved. Here, we combine molecular dynamics simulations with targeted mutagenesis to elucidate a vectorial ligand transport mechanism. Computational tunnel analysis identified two distinct high-confidence pathways: a membrane-interfacing ingress route (Channel 4/0) and a solvent-exposed egress route (Channel 3). Functional validation demonstrated that sterically occluding the ingress channel via an R364W mutation abolishes all enzymatic activity, confirming it as the obligate entry point. Conversely, rigidifying the egress channel with an engineered I205C-R239C disulfide bridge severely compromises catalytic turnover, implicating F-G loop flexibility in product release. We further provide a structural rationale for the lyase-specific deficiency caused by the V366M mutation, characterizing it as an internal steric valve. These findings establish a dual-portal model of CYP17A1 function, integrating membrane extraction with peristaltic cytosolic release, and delineate novel allosteric pockets for next-generation inhibitor design.