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

Abstract

Cytochrome P450C24A1 (CYP24A1), 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 CYP24A1 based on hybrid (2C5/ BM-3) homology modeling and affinity labeling studies clarified the role of key domains (Nterminus, A', A, and F-helices, β3a strand, & β5 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 membraneassociated domains. The most notable mutants developed by modeling were V391T and I500A, 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 (β3a strand) and Ile-500 (β5 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 3β-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 3β-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1α & 3β) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1'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 CYP24A1 has evolved a unique amino-terminal membrane § Deceased

Single A326G mutation converts human CYP24A1 from 25-OH-D 3 -24-hydroxylase into -23-hydroxylase, generating 1α,25-(OH) 2 D 3 -26,23-lactone

“…2; figure 2 in ref. 22; data not shown), we selected V79-CYP24 cells containing hCYP24A1 (7) and the opossum kidney (OK) (24) cell lines as representative models for 24-and 23-hydroxylation by CYP24A1, respectively. We then set out to optimize cell culture and incubation conditions, substrate concentrations, and chromatographic methods required to demonstrate the 24-and 23-hydroxylation pathway intermediates (Fig.…”

“…One of the important implications of this study is the growing sophistication of homology models for the vitamin D-related CYPs including CYP24A1 (21)(22)(23)25) to accurately predict substrate binding. The model (21) used here had previously defined the important roles of specific residues surrounding the substrate binding domain, these being the following: Ile-131 in A-ring and cis-triene contacts; Trp-134 and Gly-499 in substrate access; Leu-148 in side-chain contact; Met-246 in modulating regioselectivity.…”

“…These studies suggested that the two enzyme activities might have different kinetic parameters, but it was not clear whether the activities resided on a single or distinct polypeptide chains. With the cloning and expression of the recombinant protein from different species (19,20), it became clear that a single polypeptide chain was capable of both C23-and C24-hydroxylation activities (5-7).Recent studies of rat and human CYP24A1 have used homology modeling to reveal the general structure of the protein and highlight active-site residues for mutagenesis studies (21,22). Masuda et al (21) have shown the importance of residues in the BЈ-helix, BЈ/C loop, F-helix, and ␤-5 hairpin of the human enzyme.…”

“…Recent studies of rat and human CYP24A1 have used homology modeling to reveal the general structure of the protein and highlight active-site residues for mutagenesis studies (21,22). Masuda et al (21) have shown the importance of residues in the BЈ-helix, BЈ/C loop, F-helix, and ␤-5 hairpin of the human enzyme.…”

Cytochrome P450-mediated metabolism of vitamin D

“…Attempts to identify the key substrate-binding residues were originally guided by homology models (18)(19)(20)(21)(22) based upon 10-20 available crystal structures from unrelated soluble prokaryotic CYPs. Recently, the study of the active site of vitamin D-related CYPs has been further advanced by the emergence of X-ray crystallography-derived models vant vitamin D 3 -25-hydroxylase.…”