The Holo-form of the Nucleotide Binding Domain of the KdpFABC Complex from Escherichia coli Reveals a New Binding Mode

• Published in: The Journal of biological chemistry Vol. 281; no. 14; pp. 9641 - 9649
• Main Authors: Haupt, Melina, Bramkamp, Marc, Heller, Markus, Coles, Murray, Deckers-Hebestreit, Gabriele, Herkenhoff-Hesselmann, Brigitte, Altendorf, Karlheinz, Kessler, Horst
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 07.04.2006; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - chemistry; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Binding Sites; Cation Transport Proteins - chemistry; Cation Transport Proteins - metabolism; Escherichia coli; Escherichia coli - genetics; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Ion Transport - physiology; Models, Chemical; Mutation; Nuclear Magnetic Resonance, Biomolecular; Nucleotides - metabolism; Potassium - metabolism; Protein Binding; Protein Folding; Protein Structure, Tertiary; Static Electricity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M508290200

P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe377 and Lys395via a π-π stacking and a cation-π interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg317, Arg382, Asp399, and Glu348) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.