Key residues controlling binding of diverse ligands to human cytochrome P450 2A enzymes

Abstract

Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K(D)) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2'-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K(D) values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2'-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structure-based quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu(370).

Fig. 1.

UV-visible difference spectra from titration of CYP2A13 with increasing concentrations of MAP. Sequential titration additions are ordered on spectral binding image from orange to indigo. Not all spectra are shown. Inset is the nonlinear regression analysis completed using GraphPad Prism 4 from which the K_D is obtained.

Fig. 2.

Crystal structure of CYP2A13 (Protein Data Bank code 2P85) (Smith et al., 2007). The 10 mutations are highlighted in yellow. This figure was generated using PyMol (DeLano, 2003).

Fig. 3.

-Fold difference comparison of CYP2A13 mutant K_D values with CYP2A13 K_D values. Black bars represent the ligand MAP, light gray bars represent PEITC, and medium gray bars represent coumarin. CYP2A13 K_D values are all arbitrarily set to 1 to facilitate comparison of mutation effects across ligands. All mutants whose K_D varies by 3-fold or greater from CYP2A13 are shown as the mean of two independent titration experiments.

Fig. 4.

Correlation plot for pK_D (= log[K_D]) values determined via the COMBINE-based QSAR model for CYP2A6, CYP2A13, and CYP2A13 mutants relative to the experimental measurements determined herein.

Fig. 5.

Results of COMBINE analysis. A, high- and low-affinity ligands depicted in the presence of electrostatic features as gauged from COMBINE analysis. The high-scoring complex corresponds to PEITC (CPK colored sticks with green carbon atoms) in its conformation as bound to CYP2A13, and the low-scoring complex entails MAP (CPK colored sticks, with orange carbon atoms) in the conformer as bound to 2A6. The protein surface entails a composite of the largely conserved CYP2A6 and CYP2A13 structures, with features colored as follows: blue, favorable electropositive; red, favorable electronegative; cyan, unfavorable electropositive; magenta, unfavorable electronegative. B, high- and low-affinity ligands PEITC and MAP colored as in A, but in the presence of key protein steric features as gauged from COMBINE analysis. The ribbons and corresponding mesh represent the CYP2A13 parental protein (cyan ribbons and mesh) and the CYP2A13 A301G mutant protein (magenta ribbons and mesh). Voids were calculated by VOIDOO. C, conformational variation of Leu³⁷⁰ as a function of the CYP2A active site (CYP2A6, green; CYP2A13, cyan; CYP2A13/L366I, salmon; CYP2A13/G369S, blue; CYP2A13/H372R, magenta). The relative positions of bound ligands PEITC and MAP (colored as in A) are shown for reference.