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

Abstract

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1–2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

Glutamate transporters: a broad review of the most recent archaeal and human structures

“…The structure of UCPH 101 has provided important insights into the conformational changes in EAAT1 that occur upon binding of substrate and sodium ions. Furthermore, it highlights the importance of allosteric cavities that can facilitate the design of novel, selective compoundsa feature common in membrane proteins [50] (Tate, pers. comm.…”

Energy Coupling in Cation-Pumping Pyrophosphatase—Back to Mitchell

“…But this is only the first milestone in this exciting journey. Recent kinetic data (Artukka et al, 2018) suggest that active sites undergo oscillations between active and inactive conformations during catalysis, a phenomenon resembling the anchor mechanism in watches, and reflecting structural data (Vidilaseris et al, 2019) indicating asymmetrical binding of an allosteric inhibitor to two subunits. This may mean that mPPases combine two mechanisms of energy coupling-Mitchell's direct coupling and Boyer's conformational coupling (its "alternating sites" version), which were antagonists in the debate over F o F 1 -ATPase-in one protein.…”

“…An alternative possibility is that Na + and H + transport are carried out by different subunits of dimeric Na + -PPase binding Na + at a single site per subunit in a negatively cooperative manner because of dimer asymmetry (Artukka et al, 2018;Vidilaseris et al, 2019) ( Figure 1F). In this mechanism, Na + could inhibit H + transport by occupying both pump-loading sites, resembling the effect of high substrate concentration on enzymatic activity (Artukka et al, 2018).…”

Screening for <em>Thermotoga maritima</em> Membrane-Bound Pyrophosphatase Inhibitors

“…Benefits of this enzyme are for example its ease in production and purification, good thermal stability and high specific activity. TmPPase shows both high similarity in addition to the complete conservation of the position as well as identity of all catalytic residues to the protist mPPases 3,9 and to the solved structure of Vigna radiata 10 mPPase. The available structures of TmPPase in different conformations are also useful for structurebased drug design experiment (as virtual screening and de novo design).…”

“…Dissolve the compounds in dimethyl sulfoxide (DMSO) to make stock solutions of 25 100 mM in 50 200 µL, based on the availability of the compounds. NOTE: All compounds used here (Figure 2A) have been published previously 9 . If the compound solubility is low, the stock concentration can be adjusted accordingly.…”