Steroid and Protein Ligand Binding to Cytochrome P450 46A1 as Assessed by Hydrogen−Deuterium Exchange and Mass Spectrometry
- 24 March 2009
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 48 (19), 4150-4158
- https://doi.org/10.1021/bi900168m
Abstract
Cytochrome P450 46A1 (CYP46A1) is a key enzyme responsible for cholesterol elimination from the brain. This P450 can interact with different steroid substrates and protein redox partners. We utilized hydrogen−deuterium (H-D) exchange mass spectrometry for investigating CYP46A1-ligand interactions. First, we tested the applicability of the H-D exchange methodology and assessed the amide proton exchange in substrate-free and cholesterol-sulfate-bound P450. The results showed good correspondence to the available crystal structures and prompted investigation of the CYP46A1 interactions with the two steroid substrates cholesterol and 24S-hydroxycholesterol and the protein redox partner adrenodoxin (Adx). Compared to substrate-free P450, four peptides in cholesterol-bound CYP46A1 (65−80, 109−116, 151−164, and 351−361) and eight peptides in 24S-hydroxycholesterol-bound enzyme (50−64, 65−80, 109−116, 117−125, 129−143, 151−164, 260−270, and 364−373) showed altered deuterium incorporation. Most of these peptides constitute the enzyme active site, whereas the 351−361 peptide is from the region putatively interacting with the redox partner Adx. This also defines the proximal (presumably water) channel that opens in CYP46A1 upon substrate binding. Reciprocal studies of Adx binding to substrate-free and cholesterol-sulfate-bound CYP46A1 revealed changes in the deuteration of the Adx-binding site 144−150 and 351−361 peptides, active site 225−239 and 301−313 peptides, and in the 265−276 peptide, whose functional role is not yet known. The data obtained provide structural insights into how substrate and redox partner binding are coordinated and linked to the hydration of the enzyme active site.Keywords
This publication has 40 references indexed in Scilit:
- Cholesterol-metabolizing cytochromes P450: implications for cholesterol loweringExpert Opinion on Drug Metabolism & Toxicology, 2008
- Allosteric Signaling in the Biotin Repressor Occurs via Local Folding Coupled to Global Dampening of Protein DynamicsJournal of Molecular Biology, 2008
- Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brainProceedings of the National Academy of Sciences of the United States of America, 2008
- Dynamic structural changes during complement C3 activation analyzed by hydrogen/deuterium exchange mass spectrometryMolecular Immunology, 2008
- Use of complementary cation and anion heavy-atom salt derivatives to solve the structure of cytochrome P450 46A1Acta Crystallographica Section D-Biological Crystallography, 2008
- R-subunit Isoform Specificity in Protein Kinase A: Distinct Features of Protein Interfaces in PKA Types I and II by Amide H/2H Exchange Mass SpectrometryJournal of Molecular Biology, 2007
- Defining the Interacting Regions between Apomyoglobin and Lipid Membrane by Hydrogen/Deuterium Exchange Coupled to Mass SpectrometryJournal of Molecular Biology, 2007
- Mapping the Structure of Folding Cores in TIM Barrel Proteins by Hydrogen Exchange Mass Spectrometry: The Roles of Motif and Sequence for the Indole-3-glycerol Phosphate Synthase from Sulfolobus solfataricusJournal of Molecular Biology, 2007
- Amide H/2H Exchange Reveals a Mechanism of Thrombin ActivationBiochemistry, 2006
- Solvent accessibility of the thrombin-thrombomodulin interfaceJournal of Molecular Biology, 2001