Juho Kellosalo

The structure and catalytic cycle of a sodium-pumping pyrophosphatase

View of a Sodium Pump Membrane-integral pyrophosphatases (M-PPases) found in plants, protozoans, bacteria, and archaea, link pyrophosphate hydrolysis or synthesis to sodium or proton pumping and contribute to generating an electrochemical potential across the membrane. Kellosalo et al. (p. 473) report the structure of the sodium pumping M-PPase from Thermotoga maritima in the resting state with product bound. The structures reveal the conformational changes that are likely to accompany pyrophosphate binding and provide insight into the ion-pumping mechanism. Structures of a Thermotoga maritima sodium ion–pumping, membrane-integral pyrophosphatase provide a model of the pumping mechanism. 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 Asp243, Glu246, and Lys707 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.

Inorganic pyrophosphatases: One substrate, three mechanisms

Soluble inorganic pyrophosphatases (PPases) catalyse an essential reaction, the hydrolysis of pyrophosphate to inorganic phosphate. In addition, an evolutionarily ancient family of membrane‐integral pyrophosphatases couple this hydrolysis to Na+ and/or H+ pumping, and so recycle some of the free energy from the pyrophosphate. The structures of the H+‐pumping mung bean PPase and the Na+‐pumping Thermotoga maritima PPase solved last year revealed an entirely novel membrane protein containing 16 transmembrane helices. The hydrolytic centre, well above the membrane, is linked by a charged “coupling funnel” to the ionic gate about 20 Å away. By comparing the active sites, fluoride inhibition data and the various models for ion transport, we conclude that membrane‐integral PPases probably use binding of pyrophosphate to drive pumping.

Proton/sodium pumping pyrophosphatases: the last of the primary ion pumps

Membrane-bound pyrophosphatases (M-PPases) are homodimeric enzymes that couple the generation and utilization of membrane potentials to pyrophosphate (PPi) hydrolysis and synthesis. Since the discovery of the link between PPi use and proton transport in purple, non-sulphur bacteria in the 1960s, M-PPases have been found in all three domains of life and have been shown to have a crucial role in stress tolerance and in plant maturation. The discovery of sodium-pumping and sodium/proton-pumping M-PPases showed that the pumping specificity of these enzymes is not limited to protons, further suggesting that M-PPases are evolutionarily very ancient. The recent structures of two M-PPases, the Vigna radiata H(+)-pumping M-PPase and Thermotoga maritima Na(+)-pumping M-PPase, provide the basis for understanding the functional data. They show that M-PPases have a novel fold and pumping mechanism, different to the other primary pumps. This review discusses the current structural understanding of M-PPases and of ion selection among various M-PPases.

Apratoxin Kills Cells by Direct Blockade of the Sec61 Protein Translocation Channel

Apratoxin A is a cytotoxic natural product that prevents the biogenesis of secretory and membrane proteins. Biochemically, apratoxin A inhibits cotranslational translocation into the ER, but its cellular target and mechanism of action have remained controversial. Here, we demonstrate that apratoxin A prevents protein translocation by directly targeting Sec61α, the central subunit of the protein translocation channel. Mutagenesis and competitive photo-crosslinking studies indicate that apratoxin A binds to the Sec61 lateral gate in a manner that differs from cotransin, a substrate-selective Sec61 inhibitor. In contrast to cotransin, apratoxin A does not exhibit a substrate-selective inhibitory mechanism, but blocks ER translocation of all tested Sec61 clients with similar potency. Our results suggest that multiple structurally unrelated natural products have evolved to target overlapping but non-identical binding sites on Sec61, thereby producing distinct biological outcomes.

Crystallization and preliminary X-ray analysis of membrane-bound pyrophosphatases.

Abstract Membrane-bound pyrophosphatases (M-PPases) are enzymes that enhance the survival of plants, protozoans and prokaryotes in energy constraining stress conditions. These proteins use pyrophosphate, a waste product of cellular metabolism, as an energy source for sodium or proton pumping. To study the structure and function of these enzymes we have crystallized two membrane-bound pyrophosphatases recombinantly produced in Saccharomyces cerevisae: the sodium pumping enzyme of Thermotoga maritima (TmPPase) and the proton pumping enzyme of Pyrobaculum aerophilum (PaPPase). Extensive crystal optimization has allowed us to grow crystals of TmPPase that diffract to a resolution of 2.6 Å. The decisive step in this optimization was in-column detergent exchange during the two-step purification procedure. Dodecyl maltoside was used for high temperature solubilization of TmPPase and then exchanged to a series of different detergents. After extensive screening, the new detergent, octyl glucose neopentyl glycol, was found to be the optimal for TmPPase but not PaPPase.

Heterologous expression and purification of membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (M-PPases) are enzymes that couple the hydrolysis of inorganic pyrophosphate to pumping of protons or sodium ions. In plants and bacteria they are important for relieving stress caused by low energy levels during anoxia, drought, nutrient deficiency, cold and low light intensity. While they are completely absent in mammalians, they are key players in the survival of disease-causing protozoans making these proteins attractive pharmacological targets. In this work, we aimed at the purification of M-PPases in amounts suitable for crystallization as a first step to obtain structural information for drug design. We have tested the expression of eight integral membrane pyrophosphatases in Saccharomyces cerevisiae, six from bacterial and archaeal sources and two from protozoa. Two proteins originating from hyperthermophilic organisms were purified in dimeric and monodisperse active states. To generate M-PPases with an increased hydrophilic surface area, which potentially should facilitate formation of crystal contacts, phage T4 lysozyme was inserted into different extramembraneous loops of one of these M-PPases. Two of these fusion proteins were active and expressed at levels that would allow their purification for crystallization purposes.

A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity

Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.

The structure of a sodium pumping pyrophosphatase: clues to catalytic mechanism

Membrane-bound pyrophosphatases (M-PPases) couple pyrophosphate hydrolysis or synthesis to Na or H pumping. They are found in plants, bacteria and protozoans and are crucial for survival in various stress condition (such as low light intensity, anoxia, cold and mineral deficiency) and are also important for plant maturation. We have solved the metal bound resting state (TmPPase:CaMg) and product bound (TmPPase:MgPi) structures of Thermotoga maritimasodium pumping M-PPase (TmPPase) at 2.6 Å and 4 Å resolution, respectively . The resting state structure (Figure 1) shows an open active site cavity with the hydrolytic center at the top (20 Å above the membrane), followed by a “coupling funnel” formed by conserved, charged residues lining six a-helices. The “coupling funnel” ends at the gate formed by the conserved Asp243, Glu246 and Lys707 and below this is an exit channel leading to the periplasmic space. Comparison of our two TmPPase structures with the recently solved crystal structure of Vigna radiata H-pumping pyrophosphatase in the state with product analogue bound (VrPPase:PNP) shows movement of the helix 12 in VrPPase:PNP and TmPPase:MgPi (Figure 2). We presume that upon substrate binding a transient state is formed in which sliding of the helix 12 towards the periplasm/vacuolar lumen opens the gate and the exit channel and leads to ion pumping. Superimposing helices 3-6, 9-12 and 13-16 of TmPPase suggests that M-PPases arose through gene triplication.