Crystal structure of CYP24A1, a mitochondrial cytochrome P450 involved in vitamin D metabolism

Abstract

Cytochrome P450 (CYP) 24A1 catalyzes the side-chain oxidation of the hormonal form of vitamin D. Expression of CYP24A1 is up-regulated to attenuate vitamin D signaling associated with calcium homeostasis and cellular growth processes. The development of therapeutics for disorders linked to vitamin D insufficiency would be greatly facilitated by structural knowledge of CYP24A1. Here, we report the crystal structure of rat CYP24A1 at 2.5 A resolution. The structure exhibits an open cleft leading to the active-site heme prosthetic group on the distal surface that is likely to define the path of substrate access into the active site. The entrance to the cleft is flanked by conserved hydrophobic residues on helices A' and G', suggesting a mode of insertion into the inner mitochondrial membrane. A docking model for 1alpha,25-dihydroxyvitamin D(3) binding in the open form of CYP24A1 that clarifies the structural determinants of secosteroid recognition and validates the predictive power of existing homology models of CYP24A1 is proposed. Analysis of CYP24A1's proximal surface identifies the determinants of adrenodoxin recognition as a constellation of conserved residues from helices K, K'', and L that converge with an adjacent lysine-rich loop for binding the redox protein. Overall, the CYP24A1 structure provides the first template for understanding membrane insertion, substrate binding, and redox partner interaction in mitochondrial P450s.

Fig 1. The Crystal Structure of CYP24A1

A) The structure of CYP24A1 refined at 2.5 Ǻ resolution (R=0.206, R_free=0.252) shown colored by B-factor from high (red) to low (violet) temperature. P450 structural elements (α-helices, β-sheets) are labeled. Conserved residues from helices F (M245, F249), G (H271, W275, F279) and B′ (W134, Y137, R138) participate in an “aromatic cluster” centered on a water-molecule bound to R138 that promotes a membrane-directed (pw2a) substrate access channel. B) The distal surface of CYP24A1 is shown colored from negative (cyan) to positive (yellow) hydrophobicity (GES scale [41]) revealing the open channel between the A′-A helix & F-G loop regions. CHAPS molecules (pink sticks) from the crystal structure are overlaid to illustrate a model for substrate diffusion from the membrane-associated channel (pw2a) to a putative exit channel (pw3) between the D-E helix region & E-F loop [36].

Fig 2. Adaptations for Monotopic Membrane Binding

A) Interactions between CYP24A1 and the lipid-bilayer were studied computationally with the OPM server [46] and our model for membrane-binding is shown colored by hydrophobicity (GES [41]). Two membrane-insertion-sequences (MIS) are predicted for CYP24A1 that correspond to hydrophobic surface regions (helices A′ and G′) that are modeled to penetrate into the membrane's carbonyl core (∼17-22 Å) with polar lipid head groups reaching deep into the substrate-access channel between the A′-A helix and F-G loop regions. B) An orthogonal view of the OPM model illustrates the width and depth of the hydrophobic substrate access leading to the heme center. C.) Primary sequence alignments of predicted MIS-1 and D) MIS-2 binding regions demonstrate the conservation of membrane binding features across key membrane-bound P-450 forms.

Fig 3. The Active Site of Rat CYP24A1 Bound to CHAPS

A) The electron density for CHAPS (cyan carbons) (σ_A−weighted 2|F_o−F_c|-composite-omit map, 1.0σ) is shown in the heme-centered active-site of CYP24A1; important secondary structural elements (tan), CHAPS binding residues (grey carbons), the heme prosthetic group (pink carbons), and notable bond distances (Ǻ, red) are noted. Amino acid residues from the B-C loop (I131, M148) and Helices B′, F, G & I (W134, M246, F249 & T330) the K-helix/β1-4 loop (V391, F393) and the β4-1/β4-2 turn (Ile-500) mediate hydrophobic interactions with CHAPS. Polar/charged residues from the I-helix (E329), β1-4 sheet (T394, T395) and the β4-1/β4-2 turn (G499) mediated bonds to the polar face of CHAPS. The negatively charged tail of CHAPS is bound outside the active site and interacts with residues from the helices D (Y204) and F (K243).

Fig 4. Secosteroid Docking in the Open Form of Rat CYP24A1

A model for 1α,25-(OH)₂D₃ (yellow) binding in the heme-centered (pink) active-site of CYP24A1 was developed with a crystal structure-calibrated docking protocol, using Autodock 4.0 (see methods). Amino acid residues that flank the secosteroid docking site are shown from the B-B′ loop (L129, I131) B′-C loop (M148), helix F (M246, F249), helix I (L325, A326, E329, T330), the K-helix/β1-4 loop (V391, F393), the β1-4 sheet (T394, T395), the β1-3 sheet (T416), and the β4-1/β4-2 turn (G499, I500), and notable hydrogen bond distances (Ǻ, red) are given with respect to the computed H atom positions. Individual carbon atoms on 1α,25-(OH)₂D₃ are labeled for reference. Multiple hydrophobic interactions and two hydrogen bonds between the 3-OH group and the B-B′ loop (L129) and the 25-OH group and helix I (L325) are predicted to stabilize secosteroid binding in the open form. In this configuration, the C21-methyl group superimposes with a structural water (WAT-10) bound to the heme (shown as red-dotted sphere); this water molecule was excluded from the calibrated docking experiment shown here (Table S2, Fig. S5). An alternative docking model derived using control parameters, which include WAT-10, is presented in Supplemental Fig. S6.

Fig 5. Adaptations for Adrenodoxin Binding

A) CYP24A1's proximal surface is shown below the heme prosthetic group with key structural elements implicated in adrenodoxin binding noted. Basic residues from helices B, C, D, J, K and L, the Cys-Loop and the (bacterial) meander region line the positively-charged Adx binding. Fully-conserved residues from helices K (K378,K382) and L (R465,R466) known to mediate adrenodoxin binding and electron transfer in related P450s are labeled [52-55]. B) A conserved tryptophan residue (W440), from the K″ helix of mitochondrial P450s, forms a salt-bridge to the K-helix via a fully-conserved glutamate residue (E383) that may contribute to the display of the meander's lysine-rich, proximal loop, that is associated with Adx binding [57]. Residues from the L-helix (R465,R466) and Cys-loop (M462), implicated in the electron shuttle process are shown below the heme in close proximity to the K-helix. The lower portion of the active-site is also shown with CHAPS (pink) positioned above the water (WAT6) bound heme iron.