3TK3

Cytochrome P450 2B4 mutant L437A in complex with 4-(4-chlorophenyl)imidazole


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.80 Å
  • R-Value Free: 0.241 
  • R-Value Work: 0.193 
  • R-Value Observed: 0.195 

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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

Investigation by site-directed mutagenesis of the role of cytochrome P450 2B4 non-active-site residues in protein-ligand interactions based on crystal structures of the ligand-bound enzyme.

Wilderman, P.R.Gay, S.C.Jang, H.H.Zhang, Q.Stout, C.D.Halpert, J.R.

(2012) FEBS J 279: 1607-1620

  • DOI: https://doi.org/10.1111/j.1742-4658.2011.08411.x
  • Primary Citation of Related Structures:  
    3TK3

  • PubMed Abstract: 

    Residues located outside the active site of cytochromes P450 2B have exhibited importance in ligand binding, structural stability and drug metabolism. However, contributions of non-active-site residues to the plasticity of these enzymes are not known. Thus, a systematic investigation was undertaken of unique residue-residue interactions found in crystal structures of P450 2B4 in complex with 4-(4-chlorophenyl)imidazole (4-CPI), a closed conformation, or in complex with bifonazole, an expanded conformation. Nineteen mutants distributed over 11 sites were constructed, expressed in Escherichia coli and purified. Most mutants showed significantly decreased expression, especially in the case of interactions found in the 4-CPI structure. Six mutants (H172A, H172F, H172Q, L437A, E474D and E474Q) were chosen for detailed functional analysis. Among these, the K(s) of H172F for bifonazole was ∼ 20 times higher than for wild-type 2B4, and the K(s) of L437A for 4-CPI was ∼ 50 times higher than for wild-type, leading to significantly altered inhibitor selectivity. Enzyme function was tested with the substrates 7-ethoxy-4-(trifluoromethyl)coumarin, 7-methoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin (7-BR). H172F was inactive with all three substrates, and L437A did not turn over 7-BR. Furthermore, H172A, H172Q, E474D and E474Q showed large changes in k(cat)/K(M) for each of the three substrates, in some cases up to 50-fold. Concurrent molecular dynamics simulations yielded distances between some of the residues in these putative interaction pairs that are not consistent with contact. The results indicate that small changes in the protein scaffold lead to large differences in solution behavior and enzyme function.


  • Organizational Affiliation

    Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, CA 92093, USA. pwilderman@ucsd.edu


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Cytochrome P450 2B4
A, B, C, D
476Oryctolagus cuniculusMutation(s): 10 
Gene Names: CYP2B4
EC: 1.14.14.1
UniProt
Find proteins for P00178 (Oryctolagus cuniculus)
Explore P00178 
Go to UniProtKB:  P00178
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP00178
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.80 Å
  • R-Value Free: 0.241 
  • R-Value Work: 0.193 
  • R-Value Observed: 0.195 
  • Space Group: P 3
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 232.91α = 90
b = 232.91β = 90
c = 56.96γ = 120
Software Package:
Software NamePurpose
MOSFLMdata reduction
SCALAdata scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACTdata extraction
SSRLdata collection

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2011-11-16
    Type: Initial release
  • Version 1.1: 2012-05-02
    Changes: Database references
  • Version 1.2: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description