Membrane-integral Pyrophosphatases Research Articles

MPPases create a conserved anionic membrane fingerprint as identified via multi-scale simulations.

Membrane-integral pyrophosphatases (mPPases) are membrane-bound enzymes responsible for hydrolysing inorganic pyrophosphate and translocating a cation across the membrane. Their function is essential for the infectivity of clinically relevant protozoan parasites and plant maturation. Recent developments have indicated that their mechanism is more complicated than previously thought and that the membrane environment may be important for their function. In this work, we use multiscale molecular dynamics simulations to demonstrate for the first time that mPPases form specific anionic lipid interactions at 4 sites at the distal and interfacial regions of the protein. These interactions are conserved in simulations of the mPPases from Thermotoga maritima, Vigna radiata and Clostridium leptum and characterised by interactions with positive residues on helices 1, 2, 3 and 4 for the distal site, or 9, 10, 13 and 14 for the interfacial site. Due to the importance of these helices in protein stability and function, these lipid interactions may play a crucial role in the mPPase mechanism and enable future structural and functional studies.

A conserved membrane-integral pyrophosphatase anionic membrane fingerprint identified by multi-scale molecular dynamics simulations

Membrane-integral pyrophosphatases (mPPases) are clinically relevant enzymes responsible for the breakdown of inorganic pyrophosphate and translocating a cation across the membrane. Recent mechanistic developments led to the hypothesis that the protein environment may be important for function and intersubunit communication. In this study, molecular dynamics simulations at the coarse-grained and atomistic resolution of the mPPase from Thermotoga maritima (Tm-PPase) with varying lipid bilayer components were assembled and simulated.

The Function of Membrane Integral Pyrophosphatases From Whole Organism to Single Molecule.

Membrane integral pyrophosphatases (mPPases) are responsible for the hydrolysis of pyrophosphate. This enzymatic mechanism is coupled to the pumping of H+ or Na+ across membranes in a process that can be K+ dependent or independent. Understanding the movements and dynamics throughout the mPPase catalytic cycle is important, as this knowledge is essential for improving or impeding protein function. mPPases have been shown to play a crucial role in plant maturation and abiotic stress tolerance, and so have the potential to be engineered to improve plant survival, with implications for global food security. mPPases are also selectively toxic drug targets, which could be pharmacologically modulated to reduce the virulence of common human pathogens. The last few years have seen the publication of many new insights into the function and structure of mPPases. In particular, there is a new body of evidence that the catalytic cycle is more complex than originally proposed. There are structural and functional data supporting a mechanism involving half-of-the-sites reactivity, inter-subunit communication, and exit channel motions. A more advanced and in-depth understanding of mPPases has begun to be uncovered, leaving the field of research with multiple interesting avenues for further exploration and investigation.

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

As a protective envelope surrounding the bacterial cell, the peptidoglycan sacculus is a site of vulnerability and an antibiotic target. Peptidoglycan components, assembled in the cytoplasm, are shuttled across the membrane in a cycle that uses undecaprenyl-phosphate. A product of peptidoglycan synthesis, undecaprenyl-pyrophosphate, is converted to undecaprenyl-phosphate for reuse in the cycle by the membrane integral pyrophosphatase, BacA. To understand how BacA functions, we determine its crystal structure at 2.6 Å resolution. The enzyme is open to the periplasm and to the periplasmic leaflet via a pocket that extends into the membrane. Conserved residues map to the pocket where pyrophosphorolysis occurs. BacA incorporates an interdigitated inverted topology repeat, a topology type thus far only reported in transporters and channels. This unique topology raises issues regarding the ancestry of BacA, the possibility that BacA has alternate active sites on either side of the membrane and its possible function as a flippase.

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

Membrane-integral pyrophosphatases (mPPases) couple the hydrolysis of pyrophosphate (PPi) to the pumping of Na+, H+, or both these ions across a membrane. Recently solved structures of the Na+-pumping Thermotoga maritima mPPase (TmPPase) and H+-pumping Vigna radiata mPPase revealed the basis of ion selectivity between these enzymes and provided evidence for the mechanisms of substrate hydrolysis and ion-pumping. Our atomistic molecular dynamics (MD) simulations of TmPPase demonstrate that loop 5–6 is mobile in the absence of the substrate or substrate-analogue bound to the active site, explaining the lack of electron density for this loop in resting state structures. Furthermore, creating an apo model of TmPPase by removing ligands from the TmPPase:IDP:Na structure in MD simulations resulted in increased dynamics in loop 5–6, which results in this loop moving to uncover the active site, suggesting that interactions between loop 5–6 and the imidodiphosphate and its associated Mg2+ are important for holding a loop-closed conformation. We also provide further evidence for the transport-before-hydrolysis mechanism by showing that the non-hydrolyzable substrate analogue, methylene diphosphonate, induces low levels of proton pumping by VrPPase.

Integral membrane pyrophosphatases: a novel drug target for human pathogens?

Membrane-integral pyrophosphatases (mPPases) are found in several human pathogens, including Plasmodium species, the protozoan parasites that cause malaria. These enzymes hydrolyze pyrophosphate and couple this to the pumping of ions (H+ and/or Na+) across a membrane to generate an electrochemical gradient. mPPases play an important role in stress tolerance in plants, protozoan parasites, and bacteria. The solved structures of mPPases from Vigna radiata and Thermotoga maritima open the possibility of using structure-based drug design to generate novel molecules or repurpose known molecules against this enzyme. Here, we review the current state of knowledge regarding mPPases, focusing on their structure, the proposed mechanism of action, and their role in human pathogens. We also summarize different methodologies in structure-based drug design and propose an example region on the mPPase structure that can be exploited by these structure-based methods for drug targeting. Since mPPases are not found in animals and humans, this enzyme is a promising potential drug target against livestock and human pathogens.

Evolution of Vacuolar Pyrophosphatases and Vacuolar H^+-Atpases in Diatoms

To cope with changing environments and maintain optimal metabolic conditions, the control of the intracellular proton gradients has to be tightly regulated. Among the important proton pumps, vacuolar H^+-ATPases (V-ATPases) and H^+-translocating pyrophosphatases (H^+-PPases) were found to be involved in a number of physiological processes, and shown to be regulated at the expression level and to exhibit specific sub-cellular localizations. Studies of the role of these transporters are relatively scarce in algae and nearly absent in diatoms. Phylogenetic analyses disclose that diatoms, with both K^+-dependent and K^+-independent membrane integral pyrophosphatases, including proteins with high homology with a novel class of Na^+,H^+-PPases. Analyses of Phaeodactylum tricornutum EST libraries show that the gene putatively encoding a Na^+,H^+-PPase is over-expressed in urea adapted condition. Genome-wide investigations of the reservoir of V-ATPases encoding subunits demonstrate that diatoms display an expended number of genes encoding for the proteolipid subunits c of the V0 subcomplex. Preliminary localization studies show that one of V0-c subunits is associated to the endoplasmic reticulum membrane in P. tricornutum. Altogether our data highlight that the combination of comparative and functional genomic approaches reach promises to provide new information to the roles of membrane proton pumps in diatoms.

Membrane-integral pyrophosphatase subfamily capable of translocating both Na + and H +

One of the strategies used by organisms to adapt to life under conditions of short energy supply is to use the by-product pyrophosphate to support cation gradients in membranes. Transport reactions are catalyzed by membrane-integral pyrophosphatases (PPases), which are classified into two homologous subfamilies: H(+)-transporting (found in prokaryotes, protists, and plants) and Na(+)-transporting (found in prokaryotes). Transport activities have been believed to require specific machinery for each ion, in accordance with the prevailing paradigm in membrane transport. However, experiments using a fluorescent pH probe and (22)Na(+) measurements in the current study revealed that five bacterial PPases expressed in Escherichia coli have the ability to simultaneously translocate H(+) and Na(+) into inverted membrane vesicles under physiological conditions. Consistent with data from phylogenetic analyses, our results support the existence of a third, dual-specificity bacterial Na(+),H(+)-PPase subfamily, which apparently evolved from Na(+)-PPases. Interestingly, genes for Na(+),H(+)-PPase have been found in the major microbes colonizing the human gastrointestinal tract. The Na(+),H(+)-PPases require Na(+) for hydrolytic and transport activities and are further activated by K(+). Based on ionophore effects, we conclude that the Na(+) and H(+) transport reactions are electrogenic and do not result from secondary antiport effects. Sequence comparisons further disclosed four Na(+),H(+)-PPase signature residues located outside the ion conductance channel identified earlier in PPases using X-ray crystallography. Our results collectively support the emerging paradigm that both Na(+) and H(+) can be transported via the same mechanism, with switching between Na(+) and H(+) specificities requiring only subtle changes in the transporter structure.

The evolutionary history of membrane-integral pyrophosphatases supports Na+ as the ancestral coupling ion in membrane bioenergetics

The evolutionary history of membrane-integral pyrophosphatases supports Na+ as the ancestral coupling ion in membrane bioenergetics