Mechanistic analysis of the pump cycle of the P-type ATPase KdpFABC

Abstract

Due to the vital prerequisite for K+ ions in bacterial cells, involved in important processes, such as maintenance of the turgor pressure, pH homeostasis, membrane voltage and enzyme activation, E. coli comprises a set of different specialized potassium transport systems. Under K+-limiting conditions, the high affinity KdpFABC complex is expressed to sustain K+ uptake. KdpFABC is a member of the P-type ATPase family with a unique subunit composition. It consists of four subunits, and the sites of ATP hydrolysis and ion transport are well-separated on two different subunits. Only the KdpB subunit exhibits an explicit homology to other P-type ATPases and represents the catalytic subunit performing ATP hydrolysis, whereas the KdpA subunit binds and transports K+ ions and shows similarities to KcsA-like K+-channel proteins. It was demonstrated that KdpFABC, like other P-type ATPases, undergoes a reaction cycle with large conformation changes, generally represented by a so-called Post-Albers cycle. A general feature is that the ion pump toggle between two main conformational states, E1 and E2, in which the ion-binding sites alternatingly face one of both membrane sides.The aim of this work was to investigate the mechanistic aspect of the KdpFABC transport process, which would give more information about the coupling mechanism of energy-releasing ATP hydrolysis and the energy-consuming ion transport across membranes. For this purpose a fluorescence technique was employed that is based on the voltage-sensitive dye RH421, which enables a monitoring of ion movements in the membrane domain of ion-transport proteins. Based on this method, the electrogenicity of ion-binding partial reactions of the pump cycle of the detergent-solubilized KdpFABC was investigated. After finding the most appropriate detergent that preserves a functional complex, the apparent binding affinities for K+ and H+ were determined in both conformations, E1 and E2-P, and further analysis of mutual interference of K+ and H+ revealed a mixed inhibition. Binding of both K+ and H+ was found to be electrogenic. To compare transport and enzyme activity, the dependence of ATP hydrolysis on the proton concentration was measured. Furthermore, small ATPase activity was induced with Na+ and H+ in the absence of K+, supporting previous conclusion that both ion species act as weak congeners of K+. K+-binding titrations were carried out under different conditions to examine the effect of Mg2+ concentration in the electrolyte. It was found that the amount of positive charge to be bound to the membrane domain increases with the Mg2+ concentrations. This effect was assigned to the Gouy-Chapman effect.The second set of experiments was performed with the reconstituted KdpFABC in E. coli lipid vesicles, using the membrane potential indicator DiSC3(V), that allows monitoring of the electrogenic pump activity. ATP-driven K+ export across the vesicle membrane performed by the inside-out oriented KdpFABC pumps was electrogenic and confirmed that K+ is transported out of the vesicles. The experiments performed in the absence of K+ indicated an unexpected H+ translocation opposite to the well-established K+ transport, although to a significantly lower extent. The DiSC3(V) dye was used to investigate effects of different ions on the pump activity, such as H+, Mg2+, and K+. The inhibition mechanism of ADP, inorganic phosphate and o-vanadate was studied, and compared to the results reported for the Na+,K+-ATPase. Experiments on the temperature-dependence of the ATPase and pump activities were used to determine the activation energies of the respective processes.Time-resolved experiments with detergent-solubilized KdpFABC in Aminoxide WS-35 were used to analyze the kinetics of the involved processes, in the presence of different K+, H+ and ATP concentrations. These results are supportive of the proposal that the K+-binding step occurs after the phosphorylation and conformation transition reaction steps, and therefore, it has to be assigned to the dephosphorylation partial reaction.Three pump cycles derived from the general Post-Albers scheme of P-type ATPases have been introduced and discussed, with the ultimate goal to propose a possible molecular pump cycle, supported by the obtained experimental evidence. A more reliable assignment to the pump cycle of the KdpFABC requires more detailed investigation, and especially highly resolved structural information that will allow a definitive mapping of the ion-binding sites and their occupation.