This project is concerned with study of the structure and function of bovine mitochondrial, bacterial (Escherichia coli, Rhodospirillum rubrum) and Entamoeba histolytica transhydrogenases (TH), which catalyze hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to transmembrane proton translocation. As proton pumps TH are relatively simple structures, and excellent systems for study of proton translocation driven by substrate binding energy via protein conformation change. In mitochondria TH produces NADPH, which is utilized, among other things, by glutathione reductase and glutathione peroxidase to dissipate harmful H2O2 that results from dismutation of superoxide produced by the respiratory chain.
SPECIFIC AIMS are (1) Structure Studies: (a) TH is composed of 3 domains: Extramembranous domains I (400-430 residues) and III(200 residues) that bind NAD(H) and NADP(H), respectively, and membrane-intercalated domain II (360-400 residues) that carries the proton channel. We have expressed in E. coli domains I and III of bovine and R. rubrum TH. Purified domains I and III reconstitute TH activity in the absence of domain II. We have solved the crystal structure of bovine domain III containing bound NADP at 1.2 Angstrom units resolution, and have a complete data set for native crystals of R. rubrum domain I at 2.1 Angstrom units resolution. It is proposed (a) to solve the crystal structures of R. rubrum and bovine domains I, R. rubrum domain III (q nucleotides), and E. histolytica contiguous domain III-I; (b) investigate by computer modeling of these crystal structures and those of appropriate mutants the docking of domains I and III; (c) solve the crystal structure of the entire E. coli TH. (2) Mechanism of Proton Translocation: In domain II, there are 3 clusters of high sequence conservation (60-80 percent). A His and an Asp in 2 of these clusters appear to be concerned (chemically or structurally) with proton translocation. This Asp is the only conserved negatively charged residue in domain II, and its mutation to Asn or Ile also alters the affinity (Km and Kd) of TH for NADPH. The above clusters have 10 conserved hydroxylated residues, which may be involved in proton translocation. In addition, domain III contains a conserved acidic patch which may interact with positively charged residues in the cytoplasmic-side loops of domain II. It is proposed to mutate these residues in the E. coli TH to investigate their role in proton translocation and in conformational energy transfer between domains II and III. Also, docked domains I-III models will be tested by mutation of complementary interface residues and activity assays.
|Leung, Josephine H; Schurig-Briccio, Lici A; Yamaguchi, Mutsuo et al. (2015) Structural biology. Division of labor in transhydrogenase by alternating proton translocation and hydride transfer. Science 347:178-81|
|Zhao, Yonghong; Sun, Ling; Muralidhara, B K et al. (2007) Structural and thermodynamic consequences of 1-(4-chlorophenyl)imidazole binding to cytochrome P450 2B4. Biochemistry 46:11559-67|
|Smith, Brian D; Sanders, Jason L; Porubsky, Patrick R et al. (2007) Structure of the human lung cytochrome P450 2A13. J Biol Chem 282:17306-13|
|Zhao, Yonghong; White, Mark A; Muralidhara, B K et al. (2006) Structure of microsomal cytochrome P450 2B4 complexed with the antifungal drug bifonazole: insight into P450 conformational plasticity and membrane interaction. J Biol Chem 281:5973-81|
|Hunsicker-Wang, Laura M; Pacoma, Ronald L; Chen, Ying et al. (2005) A novel cryoprotection scheme for enhancing the diffraction of crystals of recombinant cytochrome ba3 oxidase from Thermus thermophilus. Acta Crystallogr D Biol Crystallogr 61:340-3|
|Sundaresan, Vidyasankar; Chartron, Justin; Yamaguchi, Mutsuo et al. (2005) Conformational diversity in NAD(H) and interacting transhydrogenase nicotinamide nucleotide binding domains. J Mol Biol 346:617-29|
|Yamaguchi, Mutsuo; Stout, C David (2003) Essential glycine in the proton channel of Escherichia coli transhydrogenase. J Biol Chem 278:45333-9|
|Sundaresan, Vidyasankar; Yamaguchi, Mutsuo; Chartron, Justin et al. (2003) Conformational change in the NADP(H) binding domain of transhydrogenase defines four states. Biochemistry 42:12143-53|
|Prasad, G Sridhar; Wahlberg, Marten; Sridhar, Vandana et al. (2002) Crystal structures of transhydrogenase domain I with and without bound NADH. Biochemistry 41:12745-54|
|Yamaguchi, Mutsuo; Stout, C David; Hatefi, Youssef (2002) The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli. J Biol Chem 277:33670-5|