ATP binding properties of the soluble part of the KdpC subunit from the Escherichia coli K(+)-transporting KdpFABC P-type ATPase.
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.
Ahnert
Franziska
F
Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, D-49069 Osnabrück, Germany.
Schmid
Roland
R
Altendorf
Karlheinz
K
Greie
Jörg-Christian
JC
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods; D017433:Protein Structure, Secondary; D012995:Solubility; D013379:Substrate Specificity
D001386:Azides; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D010446:Peptide Fragments; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli