Mechanistic Analysis of the Pump Cycle of the KdpFABC P-Type ATPase

Abstract

The high-affinity potassium uptake system KdpFABC is a unique type Ia P-type ATPase, because it separates the sites of ATP hydrolysis and ion transport on two different subunits. KdpFABC was expressed in Escherichia coli. It was then isolated and purified to homogeneity to obtain a detergent-solubilized enzyme complex that allowed the analysis of ion binding properties. The electrogenicity and binding affinities of the ion pump for K(+) and H(+) were determined in detergent-solubilized complexes by means of the electrochromic styryl dye RH421. Half-saturating K(+) concentrations and pK values for H(+) binding could be obtained in both the unphosphorylated and phosphorylated conformations of KdpFABC. The interaction of both ions with KdpFABC was studied in detail, and the presence of independent binding sites was ascertained. It is proposed that KdpFABC reconstituted in vesicles translocates protons at a low efficiency opposite from the well-established import of K(+) into the bacteria. On the basis of our results, various mechanistic pump cycle models were derived from the general Post-Albers scheme of P-type ATPases and discussed in the framework of the experimental evidence to propose a possible molecular pump cycle for KdpFABC.

The KdpFABC complex – K+transport against all odds

“…With respect to pH, steady-state K þ currents were found to be maximal at pH 7.3-7.4 (Damnjanovic et al, 2013). At lower pH, binding of protons reduced the pump current and at higher pH the release of protons inhibited the electrogenic pump activity.…”

“…The results indicate that these ions bind in an electrogenic manner to a site presumed to be in the membrane domain of the KdpFABC complex. The binding stoichiometry of each ion was relatively independent of the other ion, suggesting that there are independent sites (Damnjanovic et al, 2013). Studies of K þ -binding indicated that the relevant sites inside KdpA do not move relative to the dielectric field during phosphorylation and dephosphorylation reactions.…”

“…The initial rate of decrease can be used to quantify transport and the exponential shape reflects inhibition of transport as the potential increases. Experimental data were adopted from Damnjanovic et al (2013). Lipid vesicles are depicted as gray annuli, Kdp as a multicolored complex and DiSC3 (5) generated in strains lacking all other K þ transport systems (Buurman et al, 1995;Dorus et al, 2001).)…”

Reconstruction of the Metabolic Potential of Acidophilic Sideroxydans Strains from the Metagenome of an Microaerophilic Enrichment Culture of Acidophilic Iron-Oxidizing Bacteria from a Pilot Plant for the Treatment of Acid Mine Drainage Reveals Metabolic Versatility and Adaptation to Life at Low pH

“…Firstly, the aforementioned potassium uptake systems achieve potassium transfer into the cytoplasm only at high extracellular potassium concentrations (e.g., Damnjanovic et al, 2013). In contrast to this, three metagenomic contigs (contigs000001, contig000049, contig000055) encode an additional Kdp-type K + uptake ATPase.…”

Mechanistic principles of ion transport in the Na,K-ATPase

“…The Post-Albers scheme was found to be a general mechanism valid also for all other P-type ATPases whose pump cycles were studied so far, such as the CaATPase of the sarcoplasmic reticulum (SERCA) and the gastric H,K-ATPase [11], as well as the KdpFABC ATPase [12]. P-type ATPases share an (eponymous) phosphorylated intermediate in which the γ phosphate of the ATP molecule is transferred to a highly conserved aspartate located at the P domain of the protein (see below).…”