title: Two-dimensional NMR and all-atom molecular dynamics of cytochrome P450 CYP119 reveal hidden conformational substates.
abstract: Cytochrome P450 enzymes are versatile catalysts involved in a wide variety of biological processes from hormonal regulation and antibiotic synthesis to drug metabolism. A hallmark of their versatility is their promiscuous nature, allowing them to recognize a wide variety of chemically diverse substrates. However, the molecular details of this promiscuity have remained elusive. Here, we have utilized two-dimensional heteronuclear single quantum coherence NMR spectroscopy to examine a series of mutants site-specific labeled with the unnatural amino acid, [(13)C]p-methoxyphenylalanine, in conjunction with all-atom molecular dynamics simulations to examine substrate and inhibitor binding to CYP119, a P450 from Sulfolobus acidocaldarius. The results suggest that tight binding hydrophobic ligands tend to lock the enzyme into a single conformational substate, whereas weak binding low affinity ligands bind loosely in the active site, resulting in a distribution of localized conformers. Furthermore, the molecular dynamics simulations suggest that the ligand-free enzyme samples ligand-bound conformations of the enzyme and, therefore, that ligand binding may proceed largely through a process of conformational selection rather than induced fit.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517.
authors.forename: Jed N
authors.initials: JN
authors.lastname: Lampe
authors.affiliation: Simprota Corporation, San Francisco, California 94158.
authors.forename: Relly
authors.initials: R
authors.lastname: Brandman
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517.
authors.forename: Santhosh
authors.initials: S
authors.lastname: Sivaramakrishnan
authors.affiliation: Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94158-2517. Electronic address: ortiz@cgl.ucsf.edu.
authors.forename: Paul R Ortiz
authors.initials: PRO
authors.lastname: de Montellano
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray / Q000379:methods; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D007850:Lauric Acids / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008781:Methyltyrosines / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D011487:Protein Conformation; D013053:Spectrophotometry / Q000379:methods; D016945:Sulfolobus acidocaldarius / Q000201:enzymology*
chemical_list: D019843:Archaeal Proteins; D007850:Lauric Acids; D008024:Ligands; D008781:Methyltyrosines; C030358:lauric acid; C002350:O-methyltyrosine; D003577:Cytochrome P-450 Enzyme System; C408874:CYP119 protein, Sulfolobus solfataricus