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 pa radigm for structural understanding of cytochrome P450s, a ubiquitous prote in family with functions including the synthesis and degradation of physiol ogically 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 o pening of a substrate access channel in cytochrome P450BM-3 (P450BM-3), for P450cam such conformational changes upon substrate access have not been ob served either in different crystal structures or by standard molecular dyna mics simulations. We therefore developed a new simulation technique, the ra ndom expulsion molecular dynamics method, for probing ligand exit from buri ed active sites by imposing an artificial randomly oriented force on the li gand 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 obtain ed 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 specifi c substrate exit mechanisms, in particular the transient fluctuations and p erturbation of salt-links can be inspected directly in an animation and thu s give much better insight into possible access/exit mechanisms than static figures. In addition, center of mass traces of the substrates along differ ent expulsion pathways in P450cam can be examined interactively using MolSu rfer, a java-based protein structure viewer originally employed for navigat ing 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 a pplied for navigating along the ligand exit pathways in cytochrome P450cam, thereby permitting interactive viewing of the local protein environment si multaneously with precomputed dynamic parameters from molecular dynamics si mulations and experimentally determined crystallographic temperature factor s.