Three clusters of conformational states in p450cam reveal a multistep pathway for closing of the substrate access channel |
| |
Authors: | Lee Young-Tae Glazer Edith C Wilson Richard F Stout C David Goodin David B |
| |
Affiliation: | Department of Molecular Biology, 10550 North Torrey Pines Road, The Scripps Research Institute, La Jolla, California 92037, United States. |
| |
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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|