P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca 2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca 2+ -ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.
The disease malaria, caused by the parasite Plasmodium falciparum , remains one of the most important causes of morbidity and mortality in sub-Saharan Africa. In the absence of an efficient vaccine, the medical treatment of malaria is dependent on the use of drugs. Since artemisinin is a powerful anti-malarial drug which has been proposed to target a particular Ca 2+ -ATPase (PfATP6) in the parasite, it has been important to characterize the molecular properties of this enzyme. PfATP6 is a 139 kDa protein composed of 1228 amino acids with a 39% overall identity with rabbit SERCA1a (sarcoplasmic/endoplasmic reticulum Ca 2+ -ATPase 1a). PfATP6 conserves all sequences and motifs that are important for the function and/or structure of a SERCA, such as two high-affinity Ca 2+ -binding sites, a nucleotide-binding site and a phosphorylation site. We have been successful in isolating PfATP6 after heterologous expression in yeast and affinity chromatography in a pure, active and stable detergent-solubilized form. With this preparation, we have characterized and compared with the eukaryotic SERCA1a isoform the substrate (Ca 2+ and ATP) -dependency for PfATP6 activity as well as the specific inhibition/interaction of the protein with drugs. Our data fully confirm that PfATP6 is a SERCA, but with a distinct pharmacological profile: compared with SERCA1a, it has a lower affinity for thapsigargin and much higher affinity for cyclopiazonic acid. On the other hand, we were not able to demonstrate any inhibition by artemisinin and were also not able to monitor any binding of the drug to the isolated enzyme. Thus it is unlikely that PfATP6 plays an important role as a target for artemisinin in the parasite P. falciparum .
The sole purpose of a sperm cell is to carry genetic information from a male to a female egg. In order to accomplish this quest, the sperm cell must travel a long distance through a constantly changing environment. The success of this journey depends on membrane proteins that are uniquely expressed in sperm cells. One of these proteins is the α4 isoform of the sodium pump. This pump is optimized to cope with the ionic environment characteristic of the female reproductive tract, and its activity may be tightly coupled with secondary transporters that maintain cytoplasmic pH. Pharmacological inhibition of α4 is sufficient to inhibit sperm motility, and significant differences around the inhibitor-binding site compared with the ubiquitous α1 isoform, make α4 a feasible target in rational drug development.