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

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.