Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4.
The crystal structure of P450 2B4 bound with 1-(4-chlorophenyl)imidazole (1-CPI) has been determined to delineate the structural basis for the observed differences in binding affinity and thermodynamics relative to 4-(4-chlorophenyl)imidazole (4-CPI). Compared with the previously reported 4-CPI complex, there is a shift in the 1-CPI complex of the protein backbone in helices F and I, repositioning the side chains of Phe-206, Phe-297, and Glu-301, and leading to significant reshaping of the active site. Phe-206 and Phe-297 exchange positions, with Phe-206 becoming a ligand-contact residue, while Glu-301, rather than hydrogen bonding to the ligand, flips away from the active site and interacts with His-172. As a result the active site volume expands from 200 A3 in the 4-CPI complex to 280 A3 in the 1-CPI complex. Based on the two structures, it was predicted that a Phe-206-->Ala substitution would alter 1-CPI but not 4-CPI binding. Isothermal titration calorimetry experiments indicated that this substitution had no effect on the thermodynamic signature of 4-CPI binding to 2B4. In contrast, relative to wild-type 1-CPI binding to F206A showed significantly less favorable entropy but more favorable enthalpy. This result is consistent with loss of the aromatic side chain and possible ordering of water molecules, now able to interact with Glu-301 and exposed residues in the I-helix. Hence, thermodynamic measurements support the active site rearrangement observed in the crystal structure of the 1-CPI complex and illustrate the malleability of the active site with the fine-tuning of residue orientations and thermodynamic signatures.
Zhao
Yonghong
Y
Department of Pharmacology and Toxicology, University of Texas Medical Branch, 301 University Boulevard, Galveston, Texas 77555-1031, USA.
Sun
Ling
L
Muralidhara
B K
BK
Kumar
Santosh
S
White
Mark A
MA
Stout
C David
CD
Halpert
James R
JR
D000596:Amino Acids; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000187:drug effects; D001665:Binding Sites; D000072467:Cytochrome P450 Family 2; D017931:DNA Primers; D007093:Imidazoles / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D013816:Thermodynamics
C525744:1-(4-chlorophenyl)imidazole; D000596:Amino Acids; D017931:DNA Primers; D007093:Imidazoles; D011994:Recombinant Proteins; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)