Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains: Solution thermodynamics and molecular modeling

B. K. Muralidhara, Surendra Negi, Christopher C. Chin, Werner Braun, James R. Halpert

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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 μM, 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 μM and following the same rank order as the IC50 values. Changes in enthalpy at 25°C ranged from -6.5 to -8.8 kcal mol-1. The largest difference in binding entropy (+5.9 versus -4.1 cal mol-1K-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.

Original languageEnglish (US)
Pages (from-to)8051-8061
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number12
DOIs
StatePublished - Mar 24 2006

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ASJC Scopus subject areas

  • Biochemistry

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