A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes.Author Summary: A significant amount of information about how enzymes and other proteins function has been obtained from computer simulations. Often, the size of the system that is required to provide a sufficiently realistic model places limitations on both the timescale of the simulation, and the level of detail that can be studied. Computational approaches that utilise more than one type of method (so-called ‘multiscale methods’) allow the size of system, and timescale of study, to be increased. Membrane-bound proteins, such as cytochrome P450 enzymes (P450s), are an example of where multiscale simulations can be used. P450s are important in drug metabolism, and are known to be involved in adverse drug reactions. Access of substrate to the active site of these enzymes through the membrane is not well-understood. In the present work, a simulation pipeline is presented that leads through the construction and refinement of a realistic protein:membrane system by molecular dynamics simulations to reaction modelling. An important drug-metabolising P450, CYP3A4, is used as an example, together with the anticoagulant drug R-warfarin. Our investigations reveal that a membrane-bound model is required to fully capture the gating mechanisms and substrate ingress/egress channels. The simulation protocol described here is transferrable to other membrane-bound proteins.