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Struct Dyn. 2017 Mar 3;4(3):032105. doi: 10.1063/1.4978038. eCollection 2017 May.

Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation.

Author information

1
School of Biomedical Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds, United Kingdom.
2
Division of Pharmaceutical Chemistry and Technology, Faculty of Pharmacy, University of Helsinki , Helsinki, Finland.
3
Department of Life Sciences and Institute of Bioinformatics and Structural Biology, College of Life Sciences, National Tsing Hua University , Hsinchu 30013, Taiwan.
4
School of Physics and Astronomy and Astbury Centre for Structural Molecular Biology, University of Leeds , Leeds, United Kingdom.

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+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase 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 an apo model 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.

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