Membrane pyrophosphatases from Thermotoga maritima and Vigna radiata suggest a conserved coupling mechanism

Abstract

Membrane-bound pyrophosphatases (M-PPases), which couple proton/sodium ion transport to pyrophosphate synthesis/hydrolysis, are important in abiotic stress resistance and in the infectivity of protozoan parasites. Here, three M-PPase structures in different catalytic states show that closure of the substrate-binding pocket by helices 5–6 affects helix 13 in the dimer interface and causes helix 12 to move down. This springs a ‘molecular mousetrap', repositioning a conserved aspartate and activating the nucleophilic water. Corkscrew motion at helices 6 and 16 rearranges the key ionic gate residues and leads to ion pumping. The pumped ion is above the ion gate in one of the ion-bound structures, but below it in the other. Electrometric measurements show a single-turnover event with a non-hydrolysable inhibitor, supporting our model that ion pumping precedes hydrolysis. We propose a complete catalytic cycle for both proton and sodium-pumping M-PPases, and one that also explains the basis for ion specificity.

Insights into the mechanism of membrane pyrophosphatases by combining experiment and computer simulation

“…We used the SURFE 2 R N1, from Nanion technologies, to directly measure the movement of charge across a membrane. In the presence of PP i , VrPPase translocates protons across the membrane as expected 26 . However, in the presence of IDP, a non-hydrolyzable substrate analogue that has nitrogen in the place of the PP i central oxygen atom, the ion translocation current was lower 26 .…”

“…TmPPase has been solved in the resting state (TmPPase:Ca:Mg; PDB: 4AV3), 24 substrate analogue imidodiphosphate (IDP)-bound state (TmPPase:IDP:Na; PDB: 5LZQ), 26 phosphate analogue tungstate (WO 4 )-bound state (TmPPase:WO 4 ; PDB: 5LZR), 26 and a product-bound state (TmPPase:P i2 ; PDB: 4AV6) 24 . VrPPase has been solved in a substrate analogue imidodiphosphate (IDP)-bound state (VrPPase:IDP; PDB: 4A01) and a single phosphate-bound state (VrPPase:Pi; PDB: 5GPJ) 25 …”

Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a non-phosphorous allosteric inhibitor

“…So far, there are structures of only two mPPases: four from Thermotoga maritima (TmPPase) and two for mung bean (Vigna radiata: VrPPase). TmPPase structure has been solved in the resting state (TmPPase:Ca:Mg) 9 , with two phosphates bound (TmPPase:2P i ) 9 , with the substrate-analogue imidodiphosphate (IDP) bound (TmPPase:IDP) 10 , and with the phosphate analogue (WO 4 )-bound (TmPPase: WO 4 ) 10 . VrPPase has been solved in the IDP-bound (VrPPase:IDP) 11 and single phosphate-bound states (VrPPase:P i ) 10 .…”

“…1b). Upon binding of substrate, the active site is closed by a long loop between TMH5 and TMH6 and opened again after hydrolysis and ion pumping 9,10,11 . In the structure of TmPPase:IDP, the Na + binds within the membrane plane at the ionic gate between TMH6 and TMH16.…”

“…In the structure of TmPPase:IDP, the Na + binds within the membrane plane at the ionic gate between TMH6 and TMH16. Na + is pentacoordinated by the carboxylate groups of D243 6.50 , E246 6.53 , and D703 16.46 , the O of S247 6.54 and with the main-chain carbonyl of D243 6.50 10 (Fig. 1c).…”