title: Three clusters of conformational states in p450cam reveal a multistep pathway for closing of the substrate access channel.
abstract: Conformational changes in the substrate access channel have been observed for several forms of cytochrome P450, but the extent of conformational plasticity exhibited by a given isozyme has not been completely characterized. Here we present crystal structures of P450cam bound to a library of 12 active site probes containing a substrate analogue tethered to a variable linker. The structures provide a unique view of the range of protein conformations accessible during substrate binding. Principal component analysis of a total of 30 structures reveals three discrete clusters of conformations: closed (P450cam-C), intermediate (P450cam-I), and fully open (P450cam-O). Relative to P450cam-C, the P450cam-I state results predominantly from a retraction of helix F, while both helices F and G move in concert to reach the fully open P450cam-O state. Both P450cam-C and P450cam-I are well-defined states, while P450cam-O shows evidence of a somewhat broader distribution of conformations and includes the open form recently seen in the absence of substrate. The observed clustering of protein conformations over a wide range of ligand variants suggests a multistep closure of the enzyme around the substrate that begins by conformational selection from an ensemble of open conformations and proceeds through a well-defined intermediate, P450cam-I, before full closure to the P450cam-C state in the presence of small substrates. This multistep pathway may have significant implications for a full understanding of substrate specificity, kinetics, and coupling of substrate binding to P450 function.
authors.affiliation: Department of Molecular Biology, 10550 North Torrey Pines Road, The Scripps Research Institute, La Jolla, California 92037, United States.
authors.forename: Young-Tae
authors.initials: YT
authors.lastname: Lee
authors.forename: Edith C
authors.initials: EC
authors.lastname: Glazer
authors.forename: Richard F
authors.initials: RF
authors.lastname: Wilson
authors.forename: C David
authors.initials: CD
authors.lastname: Stout
authors.forename: David B
authors.initials: DB
authors.lastname: Goodin
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D019475:Camphor 5-Monooxygenase / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D011485:Protein Binding; D011487:Protein Conformation; D016958:Pseudomonas putida / Q000737:chemistry / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D019475:Camphor 5-Monooxygenase