Abstract
Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 A), troglitazone (2.7 A), felodipine (2.3 A), and 9-cis-retinoic acid (2.6 A) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates.
Highlights
Inate between cooperative and noncooperative models for multiple substrate binding
The glucuronide of gemfibrozil is a potent inhibitor of P450 2C8 (10, 11), and this inhibition is thought to underlie a drug-drug interaction between gemfibrozil and cerivastatin that can increase risk of rhabdomyolysis
The docking simulations suggested that anionic groups might be accommodated in a large substrate access channel located between the helix B-C loop and -sheet 1 with the potential for polar interactions with protein side chains as well as residual water molecules (14)
Summary
P450, a generic term for a cytochrome P450 enzyme (individual P450s are identified using a number-letter-number format based on amino acid sequence relatedness); CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Computer-simulated docking indicated that the large active-site cavity is likely to accommodate substrates in several possible binding poses that do not necessarily conform closely to the proposed pharmacophore. The docking simulations suggested that anionic groups might be accommodated in a large substrate access channel located between the helix B-C loop and -sheet 1 with the potential for polar interactions with protein side chains as well as residual water molecules (14). Further computer simulations indicated that all-trans-retinoic acid might bind in either a proximal site or an alternative distal site near helix BЈ that places the retinoid carboxylate close to Arg-241. The latter suggested that conformational changes could allow Arg-241 to neutralize the charge of the retinoid in the distal site. The two molecules exhibit two modes for accommodating the anionic substrates in the active site of the enzyme with the proximal substrate molecule positioned for 4-hydroxylation
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