The Structure and Catalytic Cycle of a Sodium-Pumping Pyrophosphatase

Abstract

Membrane-integral pyrophosphatases (M-PPases) are crucial for the survival of plants, bacteria, and protozoan parasites. They couple pyrophosphate hydrolysis or synthesis to Na(+) or H(+) pumping. The 2.6-angstrom structure of Thermotoga maritima M-PPase in the resting state reveals a previously unknown solution for ion pumping. The hydrolytic center, 20 angstroms above the membrane, is coupled to the gate formed by the conserved Asp(243), Glu(246), and Lys(707) by an unusual "coupling funnel" of six α helices. Comparison with our 4.0-angstrom resolution structure of the product complex suggests that helix 12 slides down upon substrate binding to open the gate by a simple binding-change mechanism. Below the gate, four helices form the exit channel. Superimposing helices 3 to 6, 9 to 12, and 13 to 16 suggests that M-PPases arose through gene triplication.

Structural basis for the reversibility of proton pyrophosphatase

“…[23][24][25] The crystal structures of the Thermotiga maritima Na C -PPase and Vigna radiata H C -PPase have been solved in 3 catalytic states -substrate-bound state (VrH C -PPase:Imidodiphosphate:Mg5 complex) (Fig. 1), resting state (TmNa C -PPase:Ca:Mg), and product-bound state (TmNa C -PPase:Pi 2 :Mg4), but the protein conformation when the ion exit channel is open is yet unavailable.…”

“…1), resting state (TmNa C -PPase:Ca:Mg), and product-bound state (TmNa C -PPase:Pi 2 :Mg4), but the protein conformation when the ion exit channel is open is yet unavailable. 23,24 The structural homology between these membrane-bound PPases is thought to underlie a common mechanism of action for these 2 proteins. 23,26 Unfortunately, the structure of sodium-bound membrane PPase is not available, and therefore the locations of the transported cation (H C or Na C ) can only be speculated.…”

“…23,24,26 Multiple hypotheses have been proposed to explain how the hydrolysis is coupled to the ion pumping activity.…”

“…27 In contrast to these 2 mechanisms that postulate that PPi hydrolysis kinetically drives proton pumping, the third mechanism, also referred to as the 'Binding Change' mechanism, conceives that release of the transported proton kinetically drives PPi hydrolysis instead. 23,26 Herein, PPi-binding induces the opening of the gate and exit channel through which the proximally bound Na C / H C ions can easily exit. The residual negative charge at this site draws a proton from the activated water and initiates PPi hydrolysis.…”

“…The residual negative charge at this site draws a proton from the activated water and initiates PPi hydrolysis. 23,26 From a mechanistic standpoint, the model proposed by Lin et al (2012) is analogous to that for bacteriorhodopsin. 23,24,26,28 Since bacteriorhodopsin uses light to generate proton gradient, using this mechanistic analogy for membrane-bound PPases would be implausible because no matter what the pH gradient, bacteriorhodopsin cannot work in reverse and generate light.…”