Crystal structure of a membrane-embedded H+-translocating pyrophosphatase

A model for the coupling of proton pumping and pyrophosphatase hydrolysis is proposed. Two types of proton-pumping protein, vacuolar H+-ATPases and H+-translocating pyrophosphatases (H+-PPases), co-exist on plant vacuolar membranes and use ATP and pyrophosphate (respectively) as energy sources for proton translocation. Whereas vacuolar H+-ATPases have been studied quite extensively, the three-dimensional structure and detailed mechanisms underlying the hydrolytic and proton-translocation reactions of H+-PPases are unclear. This paper reports the crystal structure of an H+-PPase in the presence of a non-hydrolysable substrate analogue. An unusual proton-translocation pathway is formed by six core transmembrane helices. The authors propose a model for how proton pumping and pyrophosphatase hydrolysis are coupled. H+-translocating pyrophosphatases (H+-PPases) are active proton transporters that establish a proton gradient across the endomembrane by means of pyrophosphate (PPi) hydrolysis1,2. H+-PPases are found primarily as homodimers in the vacuolar membrane of plants and the plasma membrane of several protozoa and prokaryotes2,3. The three-dimensional structure and detailed mechanisms underlying the enzymatic and proton translocation reactions of H+-PPases are unclear. Here we report the crystal structure of a Vigna radiata H+-PPase (VrH+-PPase) in complex with a non-hydrolysable substrate analogue, imidodiphosphate (IDP), at 2.35 Å resolution. Each VrH+-PPase subunit consists of an integral membrane domain formed by 16 transmembrane helices. IDP is bound in the cytosolic region of each subunit and trapped by numerous charged residues and five Mg2+ ions. A previously undescribed proton translocation pathway is formed by six core transmembrane helices. Proton pumping can be initialized by PPi hydrolysis, and H+ is then transported into the vacuolar lumen through a pathway consisting of Arg 242, Asp 294, Lys 742 and Glu 301. We propose a working model of the mechanism for the coupling between proton pumping and PPi hydrolysis by H+-PPases.