title: Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains. Solution thermodynamics and molecular modeling.
abstract: Recent x-ray structures of cytochrome P450 2B4 (CYP2B4) reveal an open form that undergoes a large-scale structural transition to a closed form upon binding to 4-(4-chlorophenyl)imidazole (4-CPI). Here, we report for the first time a complete solution thermodynamic study using isothermal titration calorimetry supported by spectroscopic studies to elucidate the conformational flexibility of CYP2B4 in binding imidazole inhibitors with different ring chemistry and side chains: 4-CPI, 1-benzylimidazole (1-BI), 1-CPI, 4-phenylimidazole (4-PI), 1-(2-(benzyloxy)ethyl)imidazole (BEI), and 1-PI. Each of the inhibitors induced type II spectral changes, and IC50 values for enzyme inhibition ranged from 0.1 to 2.4 microM, following the order 1-BI < 4-CPI < 1-CPI < 4-PI < BEI < 1-PI. Calorimetric titrations using monomeric enzyme yielded a 1:1 binding stoichiometry, with the associated KD values ranging from 0.3 to 4.8 microM and following the same rank order as the IC50 values. Changes in enthalpy at 25 degrees C ranged from -6.5 to -8.8 kcal mol(-1). The largest difference in binding entropy (+5.9 versus -4.1 cal mol(-1) K(-1)) was observed between 4-CPI and BEI, respectively, with a 2-fold difference in heat capacity changes (-604 versus -331 cal mol(-1) K(-1)), which is inferred to result from the reduction of apolar surface area of the enzyme ensuing from a conformational change upon 4-CPI binding. Accessibility to acrylamide of the only tryptophan (Trp121), which is located in helix C, was greatly decreased only in protein bound to 4-CPI. Steric restrictions hindered the perfect docking of only BEI to the closed conformation of the enzyme. The thermodynamic signature obtained for structurally similar inhibitors suggests remarkable plasticity of CYP2B4.
authors.affiliation: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston 77555, USA. bkmurali@utmb.edu
authors.forename: B K
authors.initials: BK
authors.lastname: Muralidhara
authors.forename: Surendra
authors.initials: S
authors.lastname: Negi
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Chin
authors.forename: Werner
authors.initials: W
authors.lastname: Braun
authors.forename: James R
authors.initials: JR
authors.lastname: Halpert
mesh_terms: D000178:Acrylamides / Q000737:chemistry; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry*; D001665:Binding Sites; D002151:Calorimetry; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D004305:Dose-Response Relationship, Drug; D006358:Hot Temperature; D007093:Imidazoles / Q000737:chemistry* / Q000494:pharmacology*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D015233:Models, Statistical; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013053:Spectrophotometry; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors; D014461:Ultracentrifugation
chemical_list: C510131:1-(2-(benzyloxy)ethyl)imidazole; D000178:Acrylamides; D007093:Imidazoles; D008024:Ligands; C017062:1-benzylimidazole; C033023:4(5)-phenylimidazole; C029899:imidazole; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit)