The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme's buried active site. The membrane facilitated the opening of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.

Monotopic membrane proteins bind tightly to cell membranes but do not generally span the lipid bilayer. Their interactions with lipid bilayers may be studied via coarse-grained molecular dynamics (CG-MD) simulations. Understanding such interactions is important as monotopic enzymes frequently act on hydrophobic substrates, while X-ray structures rarely provide direct information about their interactions with membranes. CG-MD self-assembly simulations enable prediction of the orientation and depth of insertion into a lipid bilayer of a monotopic protein, and also of the interactions of individual protein residues with lipid molecules. The CG-MD method has been evaluated via comparison with extended (>30 ns) atomistic simulations of monoamine oxidase, revealing good agreement between the results of coarse-grained and atomistic simulations. CG-MD simulations have been applied to a set of 11 monotopic proteins for which three-dimensional structures are available. These proteins may be divided into two groups on the basis of the results of the simulations. One group consists of those proteins which are inserted into the lipid bilayer to a limited extent, interacting mainly at the phospholipid-water interface. The second group consists of those which are inserted more deeply into the bilayer. Those monotopic proteins which are inserted more deeply cause significant local perturbation of bilayer properties such as bilayer thickness. Deeper insertion seems to correlate with a greater number of basic residues in the "foot" whereby a monotopic protein interacts with the membrane.

Monotopic proteins make up a class of membrane proteins that bind tightly to, but do not span, cell membranes. We examine and compare how two monotopic proteins, monoamine oxidase B (MAO-B) and cyclooxygenase-2 (COX-2), interact with a phospholipid bilayer using molecular dynamics simulations. Both enzymes form between three and seven hydrogen bonds with the bilayer in our simulations with basic side chains accounting for the majority of these interactions. By analyzing lipid order parameters, we show that, to a first approximation, COX-2 disrupts only the upper leaflet of the bilayer. In contrast, the top and bottom halves of the lipid tails surrounding MAO-B are more and less ordered, respectively, than in the absence of the protein. Finally, we identify which residues are important in binding individual phospholipids by counting the number and type of lipid atoms that come close to each amino acid residue. The existing models that explain how these proteins bind to bilayers were proposed following inspection of the X-ray crystallographic structures. Our results support these models and suggest that basic residues contribute significantly to the binding of these monotopic proteins to bilayers through the formation of hydrogen bonds with phospholipids.

A number of membrane-active enzymes act in a complex environment formed by the interface between a lipid bilayer and bulk water. Although x-ray diffraction studies yield structures of isolated enzyme molecules, a detailed characterization of their interactions with the interface requires a measure of how deeply such a membrane-associated protein penetrates into a lipid bilayer. Here, we apply coarse-grained (CG) molecular dynamics (MD) simulations to probe the interaction of porcine pancreatic phospholipase A2 (PLA2) with a lipid bilayer containing palmitoyl-oleoyl-phosphatidyl choline and palmitoyl-oleoyl-phosphatidyl glycerol molecules. We also used a configuration from a CG-MD trajectory to initiate two atomistic (AT) MD simulations. The results of the CG and AT simulations are evaluated by comparison with available experimental data. The membrane-binding surface of PLA2 consists of a patch of hydrophobic residues surrounded by polar and basic residues. We show this proposed footprint interacts preferentially with the anionic headgroups of the palmitoyl-oleoyl-phosphatidyl glycerol molecules. Thus, both electrostatic and hydrophobic interactions determine the location of PLA2 relative to the bilayer. From a general perspective, this study demonstrates that CG-MD simulations may be used to reveal the orientation and location of a membrane-surface-bound protein relative to a lipid bilayer, which may subsequently be refined by AT-MD simulations to probe more detailed interactions.