Scientific Report 2006

Molecular Biology

Structure and Function of Membrane-Bound Enzymes

C.D. Stout, H. Heaslet, M. Yamaguchi, V.M.M. Luna, A. Annalora, J. Chartron, V. Sundaresan

We focus on the structure and function of membrane-bound enzymes and the development of methods for crystallizing membrane proteins. We study the mechanism of transhydrogenase, a mitochondrial respiratory enzyme complex that couples proton translocation with hydride transfer. We use x-ray crystallography, biochemical and spectroscopic methods, electron microscopy studies in collaboration with M. Yeager, Department of Cell Biology, and nuclear magnetic resonance studies in collaboration with J. Dyson, Department of Molecular Biology. Crystal structures of transhydrogenase soluble domains, alone and in complex, have been determined (Fig. 1). Currently, our primary effort is to determine the structure of the intact 200-kD enzyme in its membrane-bound configuration.

Fig. 1. Superposition of 3 heterotrimers of transhydrogenase soluble domains observed in cocrystals. The presence of additional copies of the smaller soluble domain (dIII, lower right) in the crystal lattice provides a possible model for the intact enzyme in the membrane.

We are developing applications of nanodiscs for biophysical studies of integral membrane proteins in collaboration with P. Dawson, Department of Cell Biology, and S.G. Sligar, University of Illinois, Urbana-Champaign, Illinois. Nanodiscs are composed of phospholipid-binding peptides that self-assemble into discrete, water-soluble, bilayer-containing particles. Integral membrane proteins incorporated into these particles retain their enzymatic activity, are amenable to biochemical assays, and may have superior properties for crystallization in the absence of detergents. Both transhydrogenase and cytochrome ba₃ oxidase have been incorporated into nanodiscs.

In collaboration with J.A. Fee, Department of Molecular Biology, we are studying the mechanism of action of cytochrome ba₃ oxidase, the terminal enzyme of respiration. The high-resolution structure of the enzyme from Thermus thermophilus, crystallized in the presence of a detergent, has been determined. Crystallographic experiments, in concert with mutagenesis and spectroscopy, can be used to visualize intermediates in the reduction of oxygen to water and to define the paths of oxygen molecules and protons into the active site.

In collaboration with E.F. Johnson, Department of Molecular Biology; J.R. Halpert, University of Texas Medical Branch, Galveston, Texas; and others, we are characterizing structures of mammalian cytochrome P450s. These membrane-associated enzymes are involved in the biosynthesis of lipophilic hormones and specifically metabolize a remarkable diversity of exogenous compounds and drugs. More than 60 genes for P450 occur in the human genome. High-resolution structures, including substrate and inhibitor complexes, have been determined for the P450s 1A2, 2C5, 2C8, 2C9, 2A6, 2A13, 3A4, and 2B4. For 2B4, 3 structures of the enzyme in markedly different conformations provide insight to substrate binding and membrane insertion.

A major effort to determine the basis of HIV resistance to antiviral drugs is ongoing in collaboration with A.J. Olson and J.H. Elder, Department of Molecular Biology; B.E. Torbett, Department of Molecular and Experimental Medicine; and D.E. McRee, ActiveSight, San Diego, California. One aspect of this project entails determining the crystal structure of HIV protease-resistant mutants in complex with a wide range of inhibitors. Additional research projects involve crystallographic collaborations on iron-sulfur enzymes, with K.S. Carroll, University of Michigan, Ann Arbor, Michigan; electron transfer proteins, with J.A. Fee, Department of Molecular Biology; and synthetic self-assembling peptides, with M.R. Ghadiri, Department of Chemistry.

Publications

Chartron, J., Carroll, K.S., Shiau, C., Gao, H., Leary, J.A., Bertozzi, C.R., Stout, C.D. Substrate recognition, protein dynamics, and novel iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5′-phosphosulfate reductase. J. Mol. Biol. 364:152, 2006.

Heaslet, H., Kutilek, V., Morris, G.M., Lin, Y.-C., Elder, J.H., Torbett, B.E., Stout, C.D. Structural insights into the mechanisms of drug resistance in HIV-1 protease NL4-3. J. Mol. Biol. 356:967, 2006.

Hillier, B.J., Sundaresan, V., Stout, C.D., Vacquier, V.D. Expression, purification, crystallization and preliminary x-ray analysis of the olfactomedin domain from the sea urchin cell-adhesion protein amassin. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 62(Pt. 1):16, 2006.

Johnson, E.F., Stout, C.D. Structural diversity of human xenobiotic-metabolizing cytochrome P450 monooxygenases. Biochem. Biophys. Res. Commun. 338:331, 2005.

Yadav, M.K., Leman, L.J., Price, D.J., Brooks, C.L. III, Stout, C.D., Ghadiri, M.R. Coiled coils at the edge of configurational heterogeneity: structural analyses of parallel and antiparallel homotetrameric coiled coils reveal configurational sensitivity to a single solvent-exposed amino acid substitution. Biochemistry 45:4463, 2006.

Zhao, Y., White, M.A., Muralidhara, B.K., Sun, L., Halpert, J.R., Stout, C.D. Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction. J. Biol. Chem. 281:5973, 2006.