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Structure of microsomal cytochrome p4502b4 complexed with the antifungal drug bifonazole - insight into p450 conformational plasticity and membrane interaction

Academic Article
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Overview

authors

  • Zhao, Y. H.
  • White, M. A.
  • Muralidhara, B. K.
  • Sun, L.
  • Halpert, J. R.
  • Stout, C. David

publication date

  • March 2006

journal

  • Journal of Biological Chemistry  Journal

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), a modified 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 A 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 approximately 15 degrees 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.

subject areas

  • Antifungal Agents
  • Aryl Hydrocarbon Hydroxylases
  • Binding Sites
  • Cell Membrane
  • Crystallography, X-Ray
  • Imidazoles
  • Microsomes
  • Models, Molecular
  • Molecular Structure
  • Protein Binding
  • Protein Structure, Tertiary
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Identity

International Standard Serial Number (ISSN)

  • 0021-9258

Digital Object Identifier (DOI)

  • 10.1074/jbc.M511464200

PubMed ID

  • 16373351
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Additional Document Info

start page

  • 5973

end page

  • 5981

volume

  • 281

issue

  • 9

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