Membrane bound Pyrophosphatases

 

Membrane bound pyrophosphatases (mPPases) are enzymes which couple electrogenic sodium and/or proton pumping in to pyrophosphate hydrolysis. They are not present in all organisms, but are important for example in drought, cold and anoxia resistance of plants (4) or in protozoan and bacterial parasites that cause e.g. brain and stomach abscisses, malaria, Chagas disease, leishmaniasis and sleeping sickness (5). These dimeric proteins can be divided in to four functional classes: potassium-independent proton pumping enzymes and potassium-dependent proton, sodium and both proton and sodium pumping enzymes (3). TvPStructure
Figure 1 Structure of the TmPPase dimer of the resting state (side view) showing the transmembrane area (black lines) and bound Calcium- and Magnesia- ions (green spheres)
TvPCut

We are interested in studying the structure and function of mPPases. As mPPases cannot employ the rotary mechanism of F or V-ATPases and have not been found to have a similar catalytic intermediate as P-type ATPases they must employ a novel catalytic mechanism for coupling ion-pumping in to hydrolysis of a high-energy phosphoanhydride bond. Besides of being functionally interesting mPPases are also a possibly drug target for anti-bacterial and anti-protozoan drugs.

Figure 2 Cutaway view of a TmPPase monomer showing the active site of the resting-state structure. From top to bottom: hydrolytic center, coupling funnel, gate and exit channel.
Recently we solved the structure of a sodium pumping mPPase from Thermotoga maritima (TmPPase, Figure 1) at a resolution of 2.6 A (1), which is the first structure of a membrane protein solved in Finland. This structure shows that the protein has a three part active site (Figure 2) with events of the pyrophosphate hydrolysis in the hydrolytic centre linked through the coupling funnel to the opening of the gate and the exit funnel and in to ion-pumping. On the basis of the structures of the solved resting and product-bound states of TmPPase (1) and substrate bound state of Vigna radiata proton-pumping mPPase (2) we have proposed a model for the catalytic cycle of mPPases (Figure 3) in which movement of the transmembrane-helix 12 is linked to the opening of the gate and the exit channel. TvPMechanism
Figure 3 Proposed catalytic cycle of the binding-change mechanism. The numbers in the yellow bars indicare moving helices during the cycle.

References:

1) Kellosalo J, Kajander T, Kogan K, Pokharel K, Goldman A. (2012) The structure and catalytic cycle of a sodium-pumping pyrophosphatase. Science. 27;337(6093):473-6

2) Lin SM, Tsai JY, Hsiao CD, Huang YT, Chiu CL, Liu MH, Tung JY, Liu TH, Pan RL, Sun YJ. (2012) Crystal structure of a membrane-embedded H+-translocating pyrophosphatase. Nature. 484(7394):399-403.

3) Luoto HH, Baykov AA, Lahti R, Malinen AM. (2013) Membrane-integral pyrophosphatase subfamily capable of translocating both Na+ and H+. Proc Natl Acad Sci U S A .

4) Maeshima M. (2000) Vacuolar H+-pyrophosphatase. Biochim Biophys Acta. 1465(1-2):37-51

5) McIntosh MT, Vaidya AB. (2002) Vacuolar type H+ pumping pyrophosphatases of parasitic protozoa. Int J Parasitol. 32(1):1-14.