Abstract
H+-translocating pyrophosphatases (H+-PPase; EC 3.6.1.1) exists in various endomembranes of plants, bacteria, and some prokaryotes. It transports H+ into lumens at the cost of hydrolyzing the ''product of anabolic reactions'', inorganic pyrophosphate (PPi). Although the crystal structure of mung bean H+-PPase has been solved recently, the motions of H+-PPase is still unclear. Here, we applied hydrogen/deuterium exchange (HDX) coupled to mass spectrometry (MS) to monitor the dynamic of H+-PPase between the resting (apo form) and initiated states (bound with substrate analogue). When proteins were placed in a D2O solution, the backbone hydrogens, which exchange with deuterium, would be identified by MS. Accordingly, we discerned through the deuterium uptake to insight into the structural dynamic and conformational changes. HDX on the VrH+-PPase shows a significant protection againat exchange upon binding with substrate analogue, especially in the highly conserved PPi binging motif and reveals a rigid structure to form a narrow transport pathway. Additionally, Loop1, Loop5and Loop11, in the cytosolic site, exhibit a solvent inaccessible region and form a lid to cover the substrate binding pocket; N terminal and vacuolar lumen Loop12 may prevent proton back flow into the cytosol. These results highlight the more detail understanding the mechanism of VrH+-PPase and presumably for biological and agricultural interests.