title: Squeezing at entrance of proton transport pathway in proton-translocating pyrophosphatase upon substrate binding.
abstract: Homodimeric proton-translocating pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is indispensable for many organisms in maintaining organellar pH homeostasis. This unique proton pump couples the hydrolysis of PPi to proton translocation across the membrane. H(+)-PPase consists of 14-16 relatively hydrophobic transmembrane domains presumably for proton translocation and hydrophilic loops primarily embedding a catalytic site. Several highly conserved polar residues located at or near the entrance of the transport pathway in H(+)-PPase are essential for proton pumping activity. In this investigation single molecule FRET was employed to dissect the action at the pathway entrance in homodimeric Clostridium tetani H(+)-PPase upon ligand binding. The presence of the substrate analog, imidodiphosphate mediated two sites at the pathway entrance moving toward each other. Moreover, single molecule FRET analyses after the mutation at the first proton-carrying residue (Arg-169) demonstrated that conformational changes at the entrance are conceivably essential for the initial step of H(+)-PPase proton translocation. A working model is accordingly proposed to illustrate the squeeze at the entrance of the transport pathway in H(+)-PPase upon substrate binding.
authors.affiliation: Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsin Chu 30013, Taiwan.
authors.forename: Yun-Tzu
authors.initials: YT
authors.lastname: Huang
authors.forename: Tseng-Huang
authors.initials: TH
authors.lastname: Liu
authors.forename: Shih-Ming
authors.initials: SM
authors.lastname: Lin
authors.forename: Yen-Wei
authors.initials: YW
authors.lastname: Chen
authors.forename: Yih-Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.forename: Ching-Hung
authors.initials: CH
authors.lastname: Lee
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
authors.forename: Fan-Gang
authors.initials: FG
authors.lastname: Tseng
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D003017:Clostridium tetani / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D055503:Protein Multimerization / Q000502:physiology*; D011522:Protons
chemical_list: D011522:Protons; D043564:Inorganic Pyrophosphatase
keywords: Clostridium tetani Proton-translocating Pyrophosphatase; Fluorescence Resonance Energy Transfer (FRET); Membrane Enzymes; Membrane Proteins; Proton Pumps; Proton Transport