Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: Insights into substrate specificity and membrane bound structure-function

Cytochrome P450C24A1 (CYP24Al), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24Al based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A', A, and F-helices, beta 3a strand, and beta 5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains. The most notable mutants developed by modeling were V391T and 1500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D-3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (beta 3a strand) and Ile-500 (beta 5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3 beta-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)(2)D-3 analogs confirmed interactions between the 3 beta-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1 alpha and 3 beta) is obligate for high-affinity binding to rat CYP24Al Our work suggests that CYP24Al's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24Al has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring. (c) 2006 Elsevier Inc. All rights reserved.