ATP binding properties of the soluble part of the KdpC subunit from the Escherichia coli K+-transporting KdpFABC P-type ATPase

Autor(en): Ahnert, Franziska
Schmid, Roland
Altendorf, Karlheinz 
Greie, Jorg-Christian
Stichwörter: Biochemistry & Molecular Biology; CA2+-ATPASE; CALCIUM-PUMP; CIRCULAR-DICHROISM SPECTROSCOPY; COMPLEX; CRYSTAL-STRUCTURE; DOMAIN; ION-TRANSPORT; NUCLEOTIDE-BINDING; PROTEIN SECONDARY STRUCTURE; SARCOPLASMIC-RETICULUM
Erscheinungsdatum: 2006
Herausgeber: AMER CHEMICAL SOC
Journal: BIOCHEMISTRY
Volumen: 45
Ausgabe: 36
Startseite: 11038
Seitenende: 11046
Zusammenfassung: 
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 KdpCsol, 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.
ISSN: 00062960
DOI: 10.1021/bi061213p

Show full item record

Page view(s)

1
Last Week
0
Last month
1
checked on Mar 3, 2024

Google ScholarTM

Check

Altmetric