Document Detail


Peripheral ligand-binding site in cytochrome P450 3A4 located with fluorescence resonance energy transfer (FRET).
MedLine Citation:
PMID:  22194603     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The mechanisms of ligand binding and allostery in the major human drug-metabolizing enzyme cytochrome P450 3A4 (CYP3A4) were explored with fluorescence resonance energy transfer (FRET) using a laser dye, fluorol-7GA (F7GA), as a model substrate. Incorporation into the enzyme of a thiol-reactive FRET probe, pyrene iodoacetamide, allowed us to monitor the binding by FRET from the pyrene donor to the F7GA acceptor. Cooperativity of the interactions detected by FRET indicates that the enzyme possesses at least two F7GA-binding sites that have different FRET efficiencies and are therefore widely separated. To probe spatial localization of these sites, we studied FRET in a series of mutants bearing pyrene iodoacetamide at different positions, and we measured the distances from each of the sites to the donor. Our results demonstrate the presence of a high affinity binding site at the enzyme periphery. Analysis of the set of measured distances complemented with molecular modeling and docking allowed us to pinpoint the most probable peripheral site. It is located in the vicinity of residues 217-220, similar to the position of the progesterone molecule bound at the distal surface of the CYP3A4 in a prior x-ray crystal structure. Peripheral binding of F7GA causes a substantial spin shift and serves as a prerequisite for the binding in the active site. This is the first indication of functionally important ligand binding outside of the active site in cytochromes P450. The findings strongly suggest that the mechanisms of CYP3A4 cooperativity involve a conformational transition triggered by an allosteric ligand.
Authors:
Dmitri R Davydov; Jessica A O Rumfeldt; Elena V Sineva; Harshica Fernando; Nadezhda Y Davydova; James R Halpert
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Publication Detail:
Type:  Journal Article     Date:  2011-12-22
Journal Detail:
Title:  The Journal of biological chemistry     Volume:  287     ISSN:  1083-351X     ISO Abbreviation:  J. Biol. Chem.     Publication Date:  2012 Feb 
Date Detail:
Created Date:  2012-02-27     Completed Date:  2012-04-25     Revised Date:  2013-06-26    
Medline Journal Info:
Nlm Unique ID:  2985121R     Medline TA:  J Biol Chem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  6797-809     Citation Subset:  IM    
Affiliation:
Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, USA.
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MeSH Terms
Descriptor/Qualifier:
Allosteric Regulation
Binding Sites / physiology*
Catalytic Domain
Cysteine / genetics
Cytochrome P-450 CYP3A / chemistry*,  genetics
Fluorescence Resonance Energy Transfer*
Humans
Isoquinolines / chemistry*
Ligands
Models, Chemical*
Mutagenesis
Protein Structure, Secondary
Protein Structure, Tertiary
Structure-Activity Relationship
Substrate Specificity
Titrimetry
Grant Support
ID/Acronym/Agency:
R37 GM054995/GM/NIGMS NIH HHS; R37 GM054995-15/GM/NIGMS NIH HHS; R37 GM054995-16/GM/NIGMS NIH HHS; R37 GM054995-17/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/2-butyl-6-(butylamino)-1H-benzo(de)isoquinoline-1,3(2H)-dione; 0/Isoquinolines; 0/Ligands; 52-90-4/Cysteine; EC 1.14.13.67/CYP3A4 protein, human; EC 1.14.14.1/Cytochrome P-450 CYP3A
Comments/Corrections

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