Structural analysis of mammalian cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene: insight into partial enzymatic activity

Abstract

A combined structural and computational analysis of rabbit cytochrome P450 2B4 covalently bound to the mechanism-based inactivator tert-butylphenylacetylene (tBPA) has yielded insight into how the enzyme retains partial activity. Since conjugation to tBPA modifies a highly conserved active site residue, the residual activity of tBPA-labeled 2B4 observed in previous studies was puzzling. Here we describe the first crystal structures of a modified mammalian P450, which show an oxygenated metabolite of tBPA conjugated to Thr 302 of helix I. These results are consistent with previous studies that identified Thr 302 as the site of conjugation. In each structure, the core of 2B4 remains unchanged, but the arrangement of plastic regions differs. This results in one structure that is compact and closed. In this conformation, tBPA points toward helix B', making a 31° angle with the heme plane. This conformation is in agreement with previously performed in silico experiments. However, dimerization of 2B4 in the other structure, which is caused by movement of the B/C loop and helices F through G, alters the position of tBPA. In this case, tBPA lies almost parallel to the heme plane due to the presence of helix F' of the opposite monomer entering the active site to stabilize the dimer. However, docking experiments using this open form show that tBPA is able to rotate upward to give testosterone and 7-ethoxy-4-trifluoromethylcoumarin access to the heme, which could explain the previously observed partial activity.

Figure 1

Proposed mechanism for tBPA modification of 2B4. During catalytic turnover, tBPA is oxygenated to create a ketene intermediate. Nucleophilic attack by the hydroxyl group of Thr 302 forms an ester bond between 2B4 and the inhibitor. Selected bonds (red lines) indicate points of rotation to allow tBPA to change conformation once bound to the enzyme.

Figure 2

Ribbon and stick diagrams showing the active sites of both tBPA-modified 2B4 conformations. Residues found within 5 Å of tBPA make up the inactivator binding sites. In gray mesh, unbiased F_o − F_c simulated annealing omit maps contoured at 3-σ surround the Thr302-tBPA conjugates. In all three panels, the A chain of each structure is shown, which is representative of the other protein chains in the asymmetric unit. A) The active site of the closed structure surrounds tBPA with Val 104, Phe 108, Ile 114, Phe 115, Phe 206, Ile 209, Phe 297, Ala 298, Glu 301, Thr 302, and Ile 363. There is also density for a water molecule, which is also shown. Heme and tBPA are depicted as red and gray sticks, respectively and the protein is colored blue. B) Dimerization results in a different active site composition. tBPA makes contact with Arg 98, Trp121, Ser 294, Ala 298, Thr 302, Ile 363, and Val 367. From the other monomer, Val 216, Phe 217, Phe 220, Pro 221 also interact with tBPA. Heme and tBPA are colored as in A) and one half of the protein dimer is colored in green, while its symmetrical partner is in yellow. C) An overlay of the two protein conformations highlights the difference in binding modes for the Thr 302-tBPA conjugate.

Figure 3

Effects of labeling on hydrogen-deuterium exchange of 2B4. A) Difference in deuteration level of ligand-free 2B4 and tBPA-labeled 2B4 is shown with slowing in blue and acceleration in red and darker colors indicating greater magnitude of change, as indicated by the color bar. Each bar under the primary sequence is divided into rows corresponding to each time point from 10 to 100,000 s (top to bottom). B) Difference in deuteration levels after 1000 s mapped onto the closed tBPA-modified 2B4 structure. C) Difference in deuteration levels after 10,000 s mapped onto closed tBPA-modified 2B4 structure.

Figure 4

Ribbon and stick depictions of results of ligand docking into the open tBPA-modified 2B4 structure. These experiments show that a flexible Thr 302-tBPA adduct is able to rotate away from the heme to give testosterone or 7-EFC access to the heme iron. Known points of oxidation were found within 5 Å of the heme iron in several poses. These were A) carbon 16 of testosterone in five poses, B) carbon 2 of testosterone in three poses, and C) oxygen 13 of 7-EFC in four poses.