ATP Binding Properties of the Soluble Part of the KdpC Subunit from the Escherichia coli K⁺-Transporting KdpFABC P-Type ATPase

Abstract

P-Type ATPases catalyze the transport of cations across the cell envelope via site-specific hydrolysis of ATP. Modulation of enzyme activity by additional small subunits and/or a second regulatory nucleotide binding site is still a subject of discussion. In the K(+)-transporting KdpFABC complex of Escherichia coli, KdpB resembles the catalytic P-type ATPase subunit, but ATP binding also occurs in the essential but noncatalytic subunit, KdpC. For further characterization, the soluble portion of KdpC (KdpC(sol), residues Asn39-Glu190) was synthesized separately and purified to homogeneity via affinity and size exclusion chromatography. Protein integrity was confirmed by N-terminal sequencing, mass spectrometry, and circular dichroism spectroscopy, which revealed an alpha-helical content of 44% together with an 8% beta-sheet conformation consistent with the values deduced from the primary sequence. The overall protein structure was not affected by the addition of ATP to a concentration of up to 2 mM. In contrast, labeling of KdpC(sol) with the photoreactive ATP analogue 8-azido-ATP resulted in the specific incorporation of one molecule of 8-azido-ATP per peptide. No labeling could be observed upon denaturation of the protein with 0.2% sodium dodecyl sulfate, which suggests the presence of a structured nucleotide binding site. Labeling could be inhibited by preincubation with either ATP, ADP, AMP, GTP, or CTP, thus demonstrating a low specificity for nucleotides. Following 8-azido-ATP labeling and tryptic digestion of KdpC(sol), mass spectrometry showed that ATP binding occurred within the Val144-Lys161 peptide located within the C-terminal part of KdpC, thereby further demonstrating a defined nucleotide binding site. On the basis of these findings, a cooperative model in which the soluble part of KdpC activates catalysis of KdpB is suggested.

Common patterns and unique features of P-type ATPases: a comparative view on the KdpFABC complex fromEscherichia coli(Review)

“…However, in strong contrast to KdpC, these so-called FXYD proteins are not essential for enzymatic activity, thereby arguing in favor of the notion that KdpC exhibits a more unique type of regulatory function. Recently, it has been demonstrated that the isolated hydrophilic portion of KdpC (KdpC sol ) specifically binds one ATP molecule at a well-defined binding site, which is in accord with former observations that upon labeling of the KdpFABC complex with the photoreactive ATP analogue 2-azido-ATP, in addition to KdpB also KdpC was labeled significantly (Ahnert et al 2006). This argues in favor of also a regulatory function of KdpC either via interaction with the nucleotide binding pocket of KdpB or via ATP binding itself.…”

“…This argues in favor of also a regulatory function of KdpC either via interaction with the nucleotide binding pocket of KdpB or via ATP binding itself. Both the nucleotide affinity and specificity of KdpC was found to be quite low (Ahnert et al 2006), thereby rendering a cooperative regulatory mechanism together with KdpB likely. The site of ATP binding could be located within the conserved C-terminal segment of KdpC.…”

The KdpFABC complex – K+transport against all odds

“…KdpC has a single TM helix at its N-terminus and no known homologs outside of the Kdp system. The 150 residue C-terminal domain of KdpC was shown to bind and be labeled by azido-ATP with relatively low affinity (Ahnert et al, 2006;Altendorf et al, 1992). This observation led to a model in which the large C-terminal domain resided within the cytoplasm and was proposed to help the nucleotide-binding domain of KdpB bind ATP (Bramkamp et al, 2007).…”

Solution structure of the KdpFABC P-type ATPase from Escherichia coli by electron microscopic single particle analysis

“…The middle portion of the protein shows a high variability among all known KdpC polypeptides followed by the C-terminal approximately 70 amino acids, which are highly conserved. This domain is supposed to interact with the N-domain of KdpB and acts as a catalytic chaperone, which guides the ATP into the nucleotide binding pocket (Ahnert et al, 2006;Greie and Altendorf, 2007). An ab initio energy-minimized model of the 90 C-terminal amino acids was generated and fitted into the 3D map in close vicinity to the N-domain of KdpB (Figs.…”

“…One possible function is to mediate the structural and functional coupling of KdpA and KdpB, whereby KdpC strongly interacts with KdpA . Recent data on the cytosolic part of KdpC (KdpC sol ) suggest that KdpC might act as a catalytic chaperone, which cooperatively interacts with the nucleotide during catalysis and, thus, increases the rather untypically weak nucleotide binding affinity of the KdpB N-domain (Ahnert et al, 2006). Especially, glutamine 140 of KdpC might play a key role in this process, since it is part of a common ATP binding motif that could interact with the ribose moiety of the ATP molecule, which is inserted into the nucleotide binding pocket of the N-domain .…”