Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: Insight into P450 conformational plasticity and membrane interaction

Yonghong Zhao, Mark White, B. K. Muralidhara, Ling Sun, James R. Halpert, C. David Stout

Research output: Contribution to journalArticle

141 Citations (Scopus)

Abstract

To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), amodified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 Å crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent ∼15° to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.

Original languageEnglish (US)
Pages (from-to)5973-5981
Number of pages9
JournalJournal of Biological Chemistry
Volume281
Issue number9
DOIs
StatePublished - Mar 3 2006

Fingerprint

bifonazole
Plasticity
Ligands
Membranes
Catalytic Domain
Pharmaceutical Preparations
Plastics
Substrates
Burial
Azoles
Calorimetry
Antifungal Agents
Mixed Function Oxygenases
cytochrome P-450 CYP2B4 (rabbit)
Titration
Tryptophan
Conformations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole : Insight into P450 conformational plasticity and membrane interaction. / Zhao, Yonghong; White, Mark; Muralidhara, B. K.; Sun, Ling; Halpert, James R.; Stout, C. David.

In: Journal of Biological Chemistry, Vol. 281, No. 9, 03.03.2006, p. 5973-5981.

Research output: Contribution to journalArticle

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abstract = "To better understand ligand-induced structural transitions in cytochrome P450 2B4, protein-ligand interactions were investigated using a bulky inhibitor. Bifonazole, a broad spectrum antifungal agent, inhibits monooxygenase activity and induces a type II binding spectrum in 2B4dH(H226Y), amodified enzyme previously crystallized in the presence of 4-(4-chlorophenyl)imidazole (CPI). Isothermal titration calorimetry and tryptophan fluorescence quenching indicate no significant burial of protein apolar surface nor altered accessibility of Trp-121 upon bifonazole binding, in contrast to recent results with CPI. A 2.3 {\AA} crystal structure of 2B4-bifonazole reveals a novel open conformation with ligand bound in the active site, which is significantly different from either the U-shaped cleft of ligand-free 2B4 or the small active site pocket of 2B4-CPI. The O-shaped active site cleft of 2B4-bifonazole is widely open in the middle but narrow at the top. A bifonazole molecule occupies the bottom of the active site cleft, where helix I is bent ∼15° to accommodate the bulky ligand. The structure also defines unanticipated interactions between helix C residues and bifonazole, suggesting an important role of helix C in azole recognition by mammalian P450s. Comparison of the ligand-free 2B4 structure, the 2B4-CPI structure, and the 2B4-bifonazole structure identifies structurally plastic regions that undergo correlated conformational changes in response to ligand binding. The most plastic regions are putative membrane-binding motifs involved in substrate access or substrate binding. The results allow us to model the membrane-associated state of P450 and provide insight into how lipophilic substrates access the buried active site.",
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