Biology subjects

Xhaard, H.

Publications and source records attributed to Xhaard, H..

3 recordsLinked to original sources

bioRxiv2025

Expression, purification and preliminary pharmacological characterization of the Plasmodium falciparum membrane-bound pyrophosphatase type 1

Membrane-bound pyrophosphatases are integral membrane proteins that catalyze the hydrolysis of pyrophosphate into orthophosphate, while simultaneously facilitating the pumping of protons and/or sodium ions. Since mPPases are absent in humans but play a critical role in the life cycle of protist parasite, they represent promising therapeutic targets. We successfully expressed the Plasmodium falciparum type 1 mPPase in the baculovirus/insect cell expression system and purified the protein, yielding 0.3 mg per liter cell culture. Various detergents were tested for solubilization, with the protein remaining active under all selected detergents. n-dodecyl-{beta}-D-maltoside combined with cholesteryl hemisuccinate provided the highest solubility (88%). Finally, the PfPPase-VP1 was assayed against a set of fourteen antimalarial drugs, along with seven Thermotoga maritima mPPase inhibitors and fourteen compounds of unknown activity against mPPases. Only three compounds, all pyrazolo[1,5- a]pyrimidine-based TmPPase inhibitors, retained micromolar IC50 activity against PfPPase-VP1. The expression and purification of the PfPPase-VP1 will allow to conduct structural studies as well as to develop target-based screens, two steps necessary for the development of inhibitors to combat parasite disease.

biochemistry↗

bioRxiv2024

Conformational dynamics and asymmetry in multimodal inhibition of membrane-bound pyrophosphatases

Membrane-bound pyrophosphatases (mPPases) are homodimeric proteins that hydrolyse pyrophosphate and pump H+/Na+ across membranes. They are crucial for the virulence of protist pathogens, making them attractive drug targets. In this study, we investigate the inhibitory effects of seven distinct bisphosphonates against Thermotoga maritima mPPase to explore their mode of action and assist in future small molecule inhibitor development. We solved two structures of mPPase bound to the inhibitors in the enzyme active sites and probed the conformational dynamics of mPPase under multiple inhibitors and functionally relevant conditions by double electron-electron resonance (DEER) spectroscopy. We found that mPPase adopts distinct conformational equilibria in solution in the presence of different inhibitors, including states consistent with asymmetric binding in the active site (closed-open), but a symmetric apo-like conformation on the periplasmic side (open-open). Combined with solid-supported membrane-based electrophysiology recordings, this revealed that during catalysis, one monomer of the dimer remains open, and Na+ can only be pumped in a closed state. These results further support symmetry-breaking across the membrane, consistent with half-of-the-sites-reactivity.

biochemistry↗

bioRxiv2018

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

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyse 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 protist parasites, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first non-phosphorous allosteric inhibitor (Ki of 1.8 {+/-} 0.3 M) of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure at 3.7 [A] resolution together with the substrate analogue 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 {beta}-strand 1-2 during pumping, and thus preventing the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provide the first clear demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

molecular biology↗