Functional modules of KdpB, the catalytic subunit of the Kdp-ATPase from Escherichia coli

Autor(en): Bramkamp, M
Altendorf, K 
Stichwörter: ADENOSINE-TRIPHOSPHATASE; AMINO-ACID SUBSTITUTIONS; BINDING-SITE; Biochemistry & Molecular Biology; CYTOPLASMIC LOOP; FLUORESCEIN ISOTHIOCYANATE; KDPFABC COMPLEX; P-TYPE ATPASE; POTASSIUM-TRANSPORT; SARCOPLASMIC-RETICULUM; STRUCTURAL PROTEINS
Erscheinungsdatum: 2004
Herausgeber: AMER CHEMICAL SOC
Journal: BIOCHEMISTRY
Volumen: 43
Ausgabe: 38
Startseite: 12289
Seitenende: 12296
Zusammenfassung: 
The large cytoplasmic domain (H4H5) of KdpB of the KdpFABC complex (P-type ATPase) from Escherichia coli consists of two separate modules, the phosphorylation domain (KdpBP) and the nucleotide binding domain (KdpBN). The H4H5 and the KdpBN domains were purified as soluble 10Histagged fusion proteins. Both proteins exhibit a mainly (alpha-helical secondary structure as judged by CD spectroscopy. Fluorescein 5-isothiocyanate (FITC) labeling studies revealed that both proteins form a proper nucleotide binding site. Adenosine nucleotides protect the H4H5 loop but not KdpBN against FITC modification. Trinitrophenyl (TNP)-nucleotide binding studies revealed that both H4H5 and KdpBN bind nucleotides with high affinity. Furthermore, the H4H5 loop was still able to hydrolyze ATP, as well as p-nitrophenyl phosphate (pNPP). These results lend support to the notion that the separately synthesized H4H5 and KdpBN domains retain their native structure and that they reveal properties of both P2-type ATPases (e.g., Na+,K+-ATPase and Ca2+-ATPase) and P1b-type ATPases (e.g., heavy metal transporting ATPases). Furthermore, this report also emphasizes the unique position of the Kdp-ATPase within the P-type ATPase family.
ISSN: 00062960
DOI: 10.1021/bi048727d

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