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

Research output: Contribution to journalArticle

68 Citations (Scopus)

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

Fingerprint

Molecular modeling
Thermodynamics
Enzymes
Titration
Inhibitory Concentration 50
Enzyme inhibition
Calorimetry
Acrylamide
Entropy
Tryptophan
Stoichiometry
Specific heat
Plasticity
Conformations
4-(4-chlorophenyl)imidazole
cytochrome P-450 CYP2B4 (rabbit)
imidazole
Enthalpy
Hot Temperature
X-Rays

ASJC Scopus subject areas

  • Biochemistry

Cite this

Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains : Solution thermodynamics and molecular modeling. / Muralidhara, B. K.; Negi, Surendra; Chin, Christopher C.; Braun, Werner; Halpert, James R.

In: Journal of Biological Chemistry, Vol. 281, No. 12, 24.03.2006, p. 8051-8061.

Research output: Contribution to journalArticle

@article{ee5cd9dfda694f8eb1eec40893610549,
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 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.",
author = "Muralidhara, {B. K.} and Surendra Negi and Chin, {Christopher C.} and Werner Braun and Halpert, {James R.}",
year = "2006",
month = "3",
day = "24",
doi = "10.1074/jbc.M509696200",
language = "English (US)",
volume = "281",
pages = "8051--8061",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "12",

}

TY - JOUR

T1 - Conformational flexibility of mammalian cytochrome P450 2B4 in binding imidazole inhibitors with different ring chemistry and side chains

T2 - Solution thermodynamics and molecular modeling

AU - Muralidhara, B. K.

AU - Negi, Surendra

AU - Chin, Christopher C.

AU - Braun, Werner

AU - Halpert, James R.

PY - 2006/3/24

Y1 - 2006/3/24

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=33646357912&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33646357912&partnerID=8YFLogxK

U2 - 10.1074/jbc.M509696200

DO - 10.1074/jbc.M509696200

M3 - Article

VL - 281

SP - 8051

EP - 8061

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 12

ER -