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

Abstract

Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PP_i) to the pumping of Na⁺, H⁺, or both these ions across a membrane. Recently solved structures of the Na⁺-pumping <i>Thermotoga maritima</i> mPPase (TmPPase) and H⁺-pumping <i>Vigna radiata</i> 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 <i>apo</i> 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.