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

• Published in: Biochemistry (Easton) Vol. 45; no. 36; pp. 11038 - 11046
• Main Authors: Ahnert, F, Schmid, R, Altendorf, K, Greie, J-C
• Format: Journal Article
• Language: English
• Published: 12.09.2006
• Subjects: Escherichia coli
• ISSN: 0006-2960
• DOI: 10.1021/bi061213p

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 super(+)-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 sub(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 sub(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 sub(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.