title: Substrate-induced changes in domain interaction of vacuolar H⁺-pyrophosphatase.
abstract: Single molecule atomic force microscopy (smAFM) was employed to unfold transmembrane domain interactions of a unique vacuolar H(+)-pyrophosphatase (EC 3.6.1.1) from Vigna radiata. H(+)-Pyrophosphatase is a membrane-embedded homodimeric protein containing a single type of polypeptide and links PPi hydrolysis to proton translocation. Each subunit consists of 16 transmembrane domains with both ends facing the lumen side. In this investigation, H(+)-pyrophosphatase was reconstituted into the lipid bilayer in the same orientation for efficient fishing out of the membrane by smAFM. The reconstituted H(+)-pyrophosphatase in the lipid bilayer showed an authentically dimeric structure, and the size of each monomer was ∼4 nm in length, ∼2 nm in width, and ∼1 nm in protrusion height. Upon extracting the H(+)-pyrophosphatase out of the membrane, force-distance curves containing 10 peaks were obtained and assigned to distinct domains. In the presence of pyrophosphate, phosphate, and imidodiphosphate, the numbers of interaction curves were altered to 7, 8, and 10, respectively, concomitantly with significant modification in force strength. The substrate-binding residues were further replaced to verify these domain changes upon substrate binding. A working model is accordingly proposed to show the interactions between transmembrane domains of H(+)-pyrophosphatase in the presence and absence of substrate and its analog.
authors.affiliation: From the Department of Nephrology, Kidney Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan 33333.
authors.forename: Shen-Hsing
authors.initials: SH
authors.lastname: Hsu
authors.affiliation: From the Department of Nephrology, Kidney Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan 33333.
authors.forename: Yueh-Yu
authors.initials: YY
authors.lastname: Lo
authors.affiliation: the Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, and.
authors.forename: Tseng-Huang
authors.initials: TH
authors.lastname: Liu
authors.affiliation: the Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, and.
authors.forename: Yih-Jiuan
authors.initials: YJ
authors.lastname: Pan
authors.affiliation: the Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, and.
authors.forename: Yun-Tzu
authors.initials: YT
authors.lastname: Huang
authors.affiliation: the Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, and.
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
authors.affiliation: From the Department of Nephrology, Kidney Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan 33333.
authors.forename: Cheng-Chieh
authors.initials: CC
authors.lastname: Hung
authors.affiliation: Department of Engineering and System Science, College of Nuclear Science, National Tsing Hua University, Hsin Chu 30013, Taiwan.
authors.forename: Fan-Gang
authors.initials: FG
authors.lastname: Tseng
authors.affiliation: From the Department of Nephrology, Kidney Research Center, Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taoyuan 33333, cwyang@ms1.hinet.net.
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Yang
authors.affiliation: the Department of Life Science and Institute of Bioinformatics and Structural Biology, College of Life Science, and rlpan@life.nthu.edu.tw.
authors.forename: Rong-Long
authors.initials: RL
authors.lastname: Pan
mesh_terms: D007887:Fabaceae / Q000737:chemistry / Q000201:enzymology; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D017136:Ion Transport; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D018625:Microscopy, Atomic Force; D017434:Protein Structure, Tertiary; D011522:Protons; D013379:Substrate Specificity; D014617:Vacuoles / Q000201:enzymology*
chemical_list: D008051:Lipid Bilayers; D011522:Protons; D043564:Inorganic Pyrophosphatase
keywords: Atomic Force Microscopy (AFM); Bioenergetics; Force-Distance Curve; Membrane Transporter Reconstitution; Protein-Protein Interaction; Proton Pump; Vacuolar H+-pyrophosphatase