Abstract
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 10His-tagged 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 Ca(2+)-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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenosine Triphosphatases / chemistry*
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Adenosine Triphosphatases / genetics
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Adenosine Triphosphatases / isolation & purification
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Adenosine Triphosphatases / metabolism*
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Adenosine Triphosphate / metabolism
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Catalysis
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Cation Transport Proteins / chemistry*
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Cation Transport Proteins / genetics
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Cation Transport Proteins / isolation & purification
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Cation Transport Proteins / metabolism*
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Circular Dichroism
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Escherichia coli / enzymology*
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Escherichia coli / genetics
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Escherichia coli Proteins / chemistry*
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Escherichia coli Proteins / genetics
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Escherichia coli Proteins / isolation & purification
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Escherichia coli Proteins / metabolism*
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Fluorescein-5-isothiocyanate
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Models, Molecular
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Protein Structure, Quaternary
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Protein Structure, Tertiary
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Protein Subunits / chemistry*
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Protein Subunits / genetics
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Protein Subunits / isolation & purification
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Protein Subunits / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / genetics
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
Substances
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Cation Transport Proteins
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Escherichia coli Proteins
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Protein Subunits
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Recombinant Proteins
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Adenosine Triphosphate
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Adenosine Triphosphatases
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potassium translocating Kdp-ATPase, E coli
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Fluorescein-5-isothiocyanate