Substrate-binding Clusters of the K+-transporting Kdp ATPase of Escherichia coli Investigated by Amber Suppression Scanning Mutagenesis

• Published in: The Journal of biological chemistry 2001-03, Vol.276 (13), p.9590-9598
• Main Authors: Dorus, Steve, Mimura, Haruo, Epstein, Wolfgang
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - metabolism; Anions; Arginine - chemistry; Binding Sites; Carrier Proteins - metabolism; Cation Transport Proteins; Cations; Cell Division; Cell Membrane - metabolism; Codon; Dose-Response Relationship, Drug; Escherichia coli - enzymology; Escherichia coli Proteins; Histidine - chemistry; Kinetics; Lysine - chemistry; Lysine - metabolism; Models, Biological; Mutagenesis, Site-Directed; Mutation; Phenotype; Plasmids - metabolism; Potassium - metabolism; Potassium - pharmacology; Protein Binding; Protein Structure, Tertiary
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M009365200

The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K+ with high affinity and specificity. Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K+binding from the analysis of mutants with reduced affinity for K+ (Buurman, E., Kim, K.-T., and Epstein, W. (1995)J. Biol. Chem. 270, 6678–6685). K+binding by this pump has been analyzed in detail by site-directed mutagenesis. We have examined 83 of the 557 residues in KdpA, from 11 to 34 residues in each of four binding clusters known to affect K+ binding. Amber mutations were constructed in a plasmid carrying the kdpFABC structural genes. Transferring these plasmids to 12 suppressor strains, each inserting a different amino acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are important for K+ affinity but have little effect on the rate of transport. At only 21 of the residues studied did at least three substitutions alter affinity for K+, an indication that a residue is in or very near a K+ binding site. At many residues lysine was the only substitution that altered its affinity. The effect of lysine is most likely a repulsive effect of this cationic residue on K+ and thus reflects the effective distance between a residue and the site of binding or passage of K+ in KdpA. Once a crystallographic structure of Kdp is available, this measure of effective distance will help identify the path of K+ as it moves through the KdpA subunit to cross the membrane.