Scientific Report 2005

Molecular Biology

Structure and Function of Membrane-Bound Enzymes

C.D. Stout, H. Heaslet, M. Yamaguchi, V. Sundaresan, L. Hunsicker-Wang, J. Chartron

One focus of our research is the structure and function of transhydrogenase, an essential enzyme of respiration in mitochondria and bacteria. Transhydrogenase couples proton translocation across the membrane with hydride transfer between cofactors bound to soluble domains. We are determining the structure of the enzyme in its membrane-bound conformation and are studying the structures of the soluble domains. For studies of enzyme function, we are using biochemical methods and mutagenesis. Structural studies entail x-ray crystallography, electron microscopy studies done in collaboration with M. Yeager and B. Carragher, Department of Cell Biology, and nuclear magnetic resonance experiments done in collaboration with J. Dyson, Department of Molecular Biology.

In collaboration with E.F. Johnson, Department of Molecular Biology, and J.R. Halpert, University of Texas Medical Branch, Galveston, Texas, we are studying high-resolution crystal structures of mammalian cytochrome P450s. The P450s are monooxygenases involved in the biosynthesis and oxidation of lipophilic molecules, and they specifically metabolize a wide range of exogenous compounds and drugs. More than 60 genes for P450s occur in the human genome. We are studying high-resolution structures and drug-bound complexes of the human P450s 2C8, 2C9, 2A6, 3A4, and 1A2 and the rabbit P450s 2B4 and 2C5.

In collaboration with J.A. Fee, Department of Molecular Biology, we are studying the structure and mechanism of cytochrome ba₃ oxidase, the terminal enzyme of respiration responsible for the reduction of molecular oxygen to water. The high-resolution crystal structure of the enzyme from a thermophilic bacterium has been determined (Fig. 1). Crystallographic experiments, in combination with mutagenesis and spectroscopy, are being used to capture intermediates in the reaction cycle and to define the pathways of proton translocation to and from the active site within the membrane.

Fig. 1. Proposed model for sequestering the polar sugar headgroup of lipopolysaccharide (LPS) in the internal chamber of MsbA (for clarity, only 1 LPS is shown). A, LPS initially binds to the elbow helix as modeled onto the closed apo structure. B, Lipid headgroups modeled to insert into the chamber of the apo closed structure. C, As the transporter undergoes conformational changes related to binding and hydrolysis of ATP, the headgroup is “flipped” within the polar chamber while the LPS hydrocarbon chains are freely exposed and dragged through the lipid bilayer. Both LPS and MsbA conformations are modeled. D, LPS is presented to the outer leaflet of the membrane as observed in this structure. Reprinted with permission from Reyes, C.L., Chang, G. Science 308:1028, 2005.

In parallel with these studies, we are developing the application of nanodiscs for biophysical studies of integral membrane proteins. These experiments are being done in collaboration with S.G. Sligar, University of Illinois, Urbana, Illinois, and M. Yeager, Department of Cell Biology. Nanodiscs are water-soluble particles that consist of 2 copies of an engineered construct of human apolipoprotein A-I (~200 amino acids) encircling a patch of bilayer containing the approximately 160 molecules of dimyristoyl-sn-glycero-3-phosphocholine or other phospholipids. Integral membrane proteins can be inserted into these particles by spontaneous self-assembly, and to date we have incorporated both cytochrome ba₃ oxidase and transhydrogenase.

Additional research projects involve collaboration with other faculty members at Scripps Research. These projects include studies of iron-sulfur and electron transfer proteins, in collaboration with J.A. Fee and L. Noodleman, Department of Molecular Biology; RNA-protein complexes, with J.R. Williamson, Department of Molecular Biology; synthetic, self-assembling peptides, with M.R. Ghadiri, Department of Chemistry; and HIV protease inhibitor complexes, with A. Olson, Department of Molecular Biology, and B.E. Torbett, Department of Molecular and Experimental Medicine.

Publications

Carroll, K.S., Gao, H., Chen, H., Stout, C.D., Leary, J.A., Bertozzi, C.R. A conserved mechanism for sulfonucleotide reduction. PloS Biol. 3:e250, 2005.

Fee, J.A., Todaro, T.R., Luna, E., Sanders, D., Hunsicker-Wang, L.M., Patel, K.M., Bren, K.L., Gomez-Moran, E., Hill, M.G., Ai, J., Loehr, T.M., Oertling, W.A., Williams, P.A., Stout, C.D., McRee, D., Pastuszyn, A. Cytochrome rC₅₅₂, formed during expression of the truncated, Thermus thermophilus cytochrome c₅₅₂ gene in the cytoplasm of Escherichia coli, reacts spontaneously to form protein-bound, 2-formyl-4-vinyl (Spirographis) heme. Biochemistry 43:12162, 2004.

Hays, A.-M., Dunn, A.R., Chiu, R., Gray, H.B., Stout, C.D., Goodin, D.B. Conformational states of cytochrome P450cam revealed by trapping of synthetic wires. J. Mol. Biol. 344:455, 2004.

Horne, W.S., Yadav, M.K., Stout, C.D., Ghadiri, M.R. Heterocyclic peptide backbone modifications in an α-helical coiled coil. J. Am. Chem. Soc. 126:15366, 2004.

Hunsicker-Wang, L.M., Pacoma, R.L., Chen, Y., Fee, J.A., Stout, C.D. A novel cryoprotection scheme for enhancing diffraction of crystals of recombinant cytochrome ba₃ oxidase from Thermus thermophilus. Acta Crystallogr. D Biol. Crystallogr. 61:340, 2005.

Stout, C.D. Cytochrome P450 conformational diversity. Structure (Camb.) 12:1921, 2004.

Sundaresan, V., Chartron, J., Yamaguchi, M., Stout, C.D. Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains. J. Mol. Biol. 346:617, 2005.

Wester, M.R., Yano, J.K., Schoch, G.A., Yang, C., Griffin, K.J., Stout, C.D., Johnson, E.F. The structure of human cytochrome P450 2C9 complexed with flurbiprofen at 2.0-Å resolution. J. Biol. Chem. 279:35630, 2004.

Yadav, M.K., Redman, J.E., Leman, L.J., Alvarez-Gutierrez, J.M., Zhang, Y., Stout, C.D., Ghadiri, M.R. Structure-based engineering of internal cavities in coiled-coil peptides. Biochemistry 44:9723, 2005.

Yano, J.K., Hsu, M.H., Griffin, K.J., Stout, C.D., Johnson, E.F. Structures of human microsomal cytochrome P450 2A6 complexed with coumarin and methoxsalen. Nat. Struct. Mol. Biol. 12:822, 2005.

Yano, J.K., Wester, M.R., Schoch, G.A., Griffin, K.J., Stout, C.D., Johnson, E.F. The structure of human microsomal cytochrome P450 3A4 determined by x-ray crystallography to 2.05-Å resolution. J. Biol. Chem. 279:38091, 2004.