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Abstract

Cytochrome P450 enzymes (CYP, P450) play a crucial role in drug metabolism and sterol biosynthesis. CYP51 catalyzes the 14α-demethylation of sterol-like molecules. CYP51 has been shown to be a good target for antiparasitic drug design. Understanding the mechanism of P450s not only helps to invesigate drug metabolism but also to design parasite specific inhibitors that do not inhibit human orthologs. P450s are heme-containing enzymes. The active site is buried in the protein and thus, substrates need to enter the active site and products need to exit from the active site via ligand tunnels. P450s are membrane-bound proteins. They anchor in the membrane with a single transmembrane helix. For the catalytic reaction of P450s, two electrons need to be transferred from their redox partners. In eukaryotes, the most often used redox partner of P450s is cytochrome P450 reductase (CPR). CPR is also a membrane-bound protein. The association between P450 and CPR is driven by electrostatics. Current experimental evidence does not provide a full understanding of dynamics of P450s, differences between P450s, of how P450s embed in the membrane, of how ligands can enter and exit from the active site and of how P450s interact with CPR. A multiscale computational approach, including all-atom molecular dynamics simulations, coarse-grain molecular dynamics simulations and Brownian dynamics simulations was performed to tackle these problems. A model of membrane-bound T. brucei CYP51 was built and the model was shown to be consistent with existing experimental data. Simulations using models of CYP51 were compared with those of CYP2C9 and CYP2E1. The binding site residues of both T. brucei CYP51 and human CYP51 are more rigid than those of CYP2C9 and CYP2E1. Differences between the active site residues of T. brucei and human CYP51s may be key for designing T. brucei specific inhibitors. The ligand tunnels in both T. brucei and human CYP51 were also studied. Tunnel 2f serves as the predominant ligand tunnel in both proteins, but T. brucei CYP51 often uses the solvent (S) tunnel, tunnel 1 and the water (W) tunnel as the second predominant tunnel whereas human CYP51 uses tunnel 1. The difference in the use of ligand tunnels may be important for ligand specificity of the two proteins. Interactions of P450 and cytochrome P450 reductase were studied using Brownian dynamics simulations and molecular dynamics simulations. Complexes of soluble forms of different P450s, including CYP51, CYP2B4, CYP1A2, CYP2A6, CYP2C9, CYP2D6 and CYP2E1 were investigated. The P450s bind to the reductase using a similar interface, the positively charged proximal side. P450s bind to CPR with different affinities and these affinities can be inferred from the computed binding energy of the complexes. A model of the membrane-bound complex of T. brucei CYP51 and human CPR was built and simulated. This model was built using the membrane-bound model of T. brucei CYP51 and the encounter complexes of P450s and CPR generated by the Simulation of Diffusional Association (SDA 7) software, which was used for the Brownian dynamics simulation. SDA 7 is a useful software package for performing Brownian dynamics simulations of macromolecules. A webserver for SDA 7 (webSDA) was built to improve the user-friendliness of SDA 7 and automate the procedure for preparing and running SDA jobs. The webserver will not only help new users to become familiar with the SDA software but also experienced users to set up their simulations easily.