Faculty, Graduate Program
Structure and Function of Membrane Bound Enzymes, and Drug Design against HIV Protease
We use X-ray crystallography to study the structure and function of the membrane bound mitochondrial enzyme, transhydrogenase, understand the mechanism of the vitamin D metabolizing cytochrome P450, CYP24A1, and enable drug design against HIV protease. Collaborative projects focus on the mechanism of oxygen reduction by cytochrome ba3 oxidase, and the basis of substrate specificity in cytochrome P450s.
Major effort is focused on transhydrogenase (TH), a mitochondrial enzyme and large integral membrane protein. Structure determination will elucidate the mechanism by which TH transduces the proton motive force generated by respiration into the chemistry for maintaining a reducing environment. Understanding of TH function is relevant to type 2 diabetes and neurodegenerative diseases.
Mitochondrial P450 enzymes catalyze highly specific reactions in the biosynthesis and degradation of hormones. CYP24A1 oxidizes the active form vitamin D, and is an important target for inhibitor development to treat diseases linked to vitamin D insufficiency, including chronic kidney disease and cancer.
HIV protease (PR) is a proven target for drugs that suppress HIV AIDS, but treatment leads to multi-drug-resistant mutants. As a strategy to defeat the evolution of resistance, we are employing fragment based lead discovery to develop novel classes of allosteric PR inhibitors able to act in synergy with approved antiviral drugs.
In collaboration with James A. Fee at Scripps we are using X-ray crystallography to visualize key steps in the reduction of molecular oxygen to water in Thermus thermophilus cytochrome ba3 oxidase. Energy from this reaction is captured to pump protons across the inner mitochomdrial membrane, or in bacteria, the periplasmic membrane. The resulting proton gradient drives the production of ATP by ATP synthase, and the formation of NADPH by transhydrogenase.
Ph.D., Biology, University of Wisconsin-Madison, 1976
Annalora, A., Goodin, D.B., Hong, W.-X., Zhang, Q., Johnson, E.F., Stout, C.D. (2010) Crystal structure of CYP24A1, a Mitochondrial Cytochrome P450 involved in vitamin D metabolism. J. Mol. Biol. 396, 441-451.
Gay, S.C., Shah, M.B., Talakad, J.C., Maekawa, K., Roberts, A.G., Wilderman, P.R., Sun, L., Yang, J.Y., Huelga, S.C., Zhang, Q., Stout, C.D., Halpert, J.R. (2010) Crystal Structure of a Cytochrome P450 2B6 Genetic Variant in Complex with the Inhibitor 4-(4-Chlorophenyl)imidazole at 2.0 Å Resolution. Mol. Pharmacol. 77, 529-538.
Perryman, A.L., Zhang, Q., Soutter, H.H., Rosenfeld, R., McRee, D.E., Olson, A.J., Elder, J.E., Stout, C.D. (2010) Fragment-Based Screen against HIV Protease. Chem. Biol. & Drug Design 75, 257-268.
Mast, N., Charvet, C., Pikuleva, I., Stout, C.D. (2010) Structural basis of drug binding to CYP46A1, an enzyme that controls cholesterol turnover in the brain. J. Biol. Chem. 285, 31783-31795.
Wilderman, P.R., Shah, M.B., Liu, T., Li, S., Hsu, S., Roberts, A.G., Goodlett, D.R., Woods, V.L., Stout, C.D., Halpert, J.R. (2010) Plasticity of Cytochrome P450 2B4 as Investigated by Hydrogen-Deuterium Exchange Mass Spectrometry and X-Ray Crystallography. J. Biol. Chem. 285, 38602-38611.
Gay, S.C., Roberts, A., Maekawa, K., Talakad, J., Hong, W.-X., Zhang, Q., Stout, C.D., Halpert, J.R. (2010) Structures of Cytochrome P450 2B4 Complexed with the Antiplatelet Drugs Ticlopidine and Clopidogrel. Biochemistry 49, 8709-8720.
Mast, N., Annalora, A.J., Lodowski, D., Palczewski, K., Stout, C.D., Pikuleva, I. (2010) Structural basis for three-step sequential catalysis by the cholesterol side chain cleavage enzyme CYP11A1. J Biol. Chem., December 15, 2010, doi:10.1074/jbc.M110.188433.
Wang, A., Savas, U., Stout, C.D., Johnson, E.F. (2010) Structural characterization of the complex between [alpha]-naphthoflavone and human cytochrome P450 1B1. J. Biol. Chem., December 8, 2010, doi:10.1074/jbc.M110.204420.