Roles of the Hydrophobic Gate and Exit Channel in Vigna radiata Pyrophosphatase Ion Translocation

Abstract

Membrane-embedded pyrophosphatase (M-PPase) hydrolyzes pyrophosphate to drive ion (H+ and/or Na+) translocation. We determined crystal structures and functions of Vigna radiata M-PPase (VrH+-PPase), the VrH+-PPase–2Pi complex and mutants at hydrophobic gate (residue L555) and exit channel (residues T228 and E225). Ion pore diameters along the translocation pathway of three VrH+-PPases complexes (Pi-, 2Pi- and imidodiphosphate-bound states) present a unique wave-like profile, with different pore diameters at the hydrophobic gate and exit channel, indicating that the ligands induced pore size alterations. The 2Pi-bound state with the largest pore diameter might mimic the hydrophobic gate open. In mutant structures, ordered waters detected at the hydrophobic gate among VrH+-PPase imply the possibility of solvation, and numerous waters at the exit channel might signify an open channel. A salt-bridge, E225–R562 is at the way out of the exit channel of VrH+-PPase; E225A mutant makes the interaction eliminated and reveals a decreased pumping ability. E225–R562 might act as a latch to regulate proton release. A water wire from the ion gate (R-D-K-E) through the hydrophobic gate and into the exit channel may reflect the path of proton transfer.