title: Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation.
abstract: Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumpingThermotoga maritimamPPase (TmPPase) and H+-pumpingVigna radiatamPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5-6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating anapomodel of TmPPase by removing ligands from the TmPPase:IDP:Na structure in MD simulations resulted in increased dynamics in loop 5-6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5-6 and the imidodiphosphate and its associated Mg2+are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase.
authors.affiliation: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds, United Kingdom.
authors.forename: Nita R
authors.initials: NR
authors.lastname: Shah
authors.affiliation: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds, United Kingdom.
authors.forename: Craig
authors.initials: C
authors.lastname: Wilkinson
authors.affiliation: School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds, United Kingdom.
authors.forename: Steven P D
authors.initials: SP
authors.lastname: Harborne
authors.affiliation: Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki , Helsinki, Finland.
authors.forename: Ainoleena
authors.initials: A
authors.lastname: Turku
authors.affiliation: Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Kun-Mou
authors.initials: KM
authors.lastname: Li
authors.affiliation: Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Yuh-Ju
authors.initials: YJ
authors.lastname: Sun
authors.affiliation: School of Physics and Astronomy and Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds, United Kingdom.
authors.forename: Sarah
authors.initials: S
authors.lastname: Harris
authors.forename: Adrian
authors.initials: A
authors.lastname: Goldman