Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active-site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme

Abstract

Residues located outside the active site of cytochromes P450 2B have exhibited importance in ligand binding, structural stability and drug metabolism. However, contributions of non-active-site residues to the plasticity of these enzymes are not known. Thus, a systematic investigation was undertaken of unique residue-residue interactions found in crystal structures of P450 2B4 in complex with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex with bifonazole, an expanded conformation. Nineteen mutants distributed over 11 sites were constructed, expressed in Escherichia coli and purified. Most mutants showed significantly decreased expression, especially in the case of interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q, L437A, E474D and E474Q) were chosen for detailed functional analysis. Among these, the K(s) of H172F for bifonazole was ∼ 20 times higher than for wild-type 2B4, and the K(s) of L437A for 4-CPI was ∼ 50 times higher than for wild-type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin, 7-methoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin (7-BR). H172F was inactive with all three substrates, and L437A did not turn over 7-BR. Furthermore, H172A, H172Q, E474D and E474Q showed large changes in k(cat)/K(M) for each of the three substrates, in some cases up to 50-fold. Concurrent molecular dynamics simulations yielded distances between some of the residues in these putative interaction pairs that are not consistent with contact. The results indicate that small changes in the protein scaffold lead to large differences in solution behavior and enzyme function.

Figure 1

Ribbon diagram showing the location of known SNPs (yellow sphere and sticks) in 2B6. The heme is shown as red sticks. All the known coding sequence variants of 2B6 are located outside of the active site.

Figure 2

Location of interactions interrupted by mutation. A) Interactions in the 2B4-4-CPI structure (1SUO). B) Interactions in the 2B4-bifonazole structure (2BDM). Residues in each interaction are shown as sticks with a red sphere for the alpha carbon. Positions for focused analysis from SNP sites (H172A/F/Q), the 2B4-4-CPI structure (L437A), and the 2B4-bifonazole structure (E474D/Q) are marked with an asterisk (*).

Figure 3

Stability of 2B4 wild type and mutant proteins. A) Thermal melting temperature. B) Thermal inactivation rate constant. C) Hydrogen peroxide mediated heme depletion rate constant. Black bars represent P450 inactivation; gray bars represent P420 inactivation.

Figure 4

Spectral binding of imidazole ligands to 2B4 wild type and mutant proteins. A) K_s for 4-CPI. B) K_s for bifonazole. C) Ratio of constants for 4-CPI to bifonazole.

Figure 5

Molecular dynamics simulation of 2B4 wild type. A) Representative change in backbone RMSD over 2 ns simulation for wild type protein using the 4-CPI structure (1PO5) as the starting structure; this change is representative of changes found in simulations of mutant proteins or in the simulation using the bifonazole structure (2BDM) as the starting structure. B) Distance between closest atoms for each interaction found in the crystal structures of 2B4 complexed with bifonazole or 4-CPI for mutants interrupting interactions from the respective structure. The five interactions from the bifonazole structure are listed at the top, and the four interactions from the 4-CPI structure are on the bottom. C) Distance between residues found in the proposed hydrogen bonding network in cytochromes 2B from Gay et al. [31].

Figure 6

Overlay of wild type and mutant 2B4 in complex with 4-CPI. A) Overlay of C_α backbone of wild type 2B4 (yellow, PDB ID: 1SUO) and the L437A mutant (blue, PDB ID: 3TK3). B) The active site and heme support of wild type 2B4 and the L437A mutant.