Molecular dynamics simulations of P450BM3 - Examination of substrate-induced conformational change

Cytochrome P450 BM3, of bacterial origin, is one of only five isozymes of t he ubiquitous family of over 400 metabolizing heme proteins with a known cr ystal structure and only one of two with both substrate-free and substrate- bound forms determined. P450 BM3 is of particular interest since it has a s imilar function and similar substrates as mammalian P450s particularly of t he 4A subfamily. Thus, the extent to which the substrate-free form of P450 BM3 undergoes a conformational change upon binding of a typical fatty acid substrate, palmitoleic acid, has been the subject of recent active experime ntal effort. Surprisingly, direct examination of the substrate-free (pdb2hp d.ent and pdb2bmh.ent) and substrate-bound (pdb1fag.ent) forms do not provi de a clear answer to this question. The main reason for this ambiguity is t hat the two substrate-free monomers reported in the crystal structures them selves have significantly different conformations from each other, one with a more open substrate-access channel than the other. Since there is no way to tell to which substrate-free form the substrate binds, the effect of su bstrate binding cannot be deduced directly from comparisons of the experime ntal substrate-bound and substrate-free forms. The computational studies re ported here have been designed to more robustly establish the effect of sub strate binding on this isozyme. Specifically, molecular dynamics simulation s were performed for each of the two substrate-free forms found in the asym metric unit of the X-ray structure and for the two corresponding substrate- bound forms, constructed by docking palmitloeic acid into each of them. Com parisons of the results showed that palmitoleic acid binding had little eff ect on the conformation of the more closed substrate-free form of P450 BM3. By contrast, in the more open substrate-free form, this same substrate ind uced a closing of the entrance to the substrate-binding channel. The MD ave raged structure of these two complexes obtained from docking of pamitoleic acid into the two asymmetric units of the substrate-free form were also com pared to that obtained starting with the X-ray structure of the substrate-b ound form. These results taken together led to the conclusion that, if inde ed the substrate induces conformational changes in P450 BM3, the mouth of t he substrate-access channel first closes down in response to the presence o f the substrate, followed by rotation of the F-G domain to further optimize the P450 EMS-substrate interaction that would occur at a later stage.