Substrate access to cytochrome P450cam investigated by molecular dynamics simulations:: An interactive look at the underlying mechanisms

Cytochrome P450cam (P450cam) from Pseudomonas putida has long provided a paradigm for structural understanding of cytochrome P450s, a ubiquitous protein family with functions including the synthesis and degradation of physiologically important compounds, e.g. steroids and prostaglandins, and of many xenobiotics, e.g. drugs and procarcinogens. The mechanism by which camphor, the natural substrate of P450cam, accesses the buried active site is not clear. While there is recent crystallographic and simulation evidence for opening of a substrate access channel in cytochrome P450BM-3 (P450BM-3), for P450cam such conformational changes upon substrate access have not been observed either in different crystal structures or by standard molecular dynamics simulations. We therefore developed a new simulation technique, the random expulsion molecular dynamics method, for probing ligand exit from buried active sites by imposing an artificial randomly oriented force on the ligand in addition to the standard molecular dynamics force field (Ludemann, S. K., Lounnas, V. and Wade, R. C., J.. Biol., 303, 797-811, 2000). In the present paper, representative animations for the three exit pathways obtained by the random expulsion molecular dynamics method are shown. The method was tested in simulations of the substrate-bound structure of P450BM-3, for which an animation is also shown. The protein dynamics involved in specific substrate exit mechanisms, in particular the transient fluctuations and perturbation of salt-links can be inspected directly in an animation and thus give much better insight into possible access/exit mechanisms than static figures. In addition, center of mass traces of the substrates along different expulsion pathways in P450cam can be examined interactively using MolSurfer, a java-based protein structure viewer originally employed for navigating molecular interfaces (Gabdoulline, R. R., Wade, R. C. and Walther, D., Trends Biochem. Sci 24, 285-7 (1999)). In the present paper, this tool is applied for navigating along the ligand exit pathways in cytochrome P450cam, thereby permitting interactive viewing of the local protein environment simultaneously with precomputed dynamic parameters from molecular dynamics simulations and experimentally determined crystallographic temperature factors.