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 acc umulates K+ with high affinity and specificity, Residues clustered in four regions of the KdpA subunit of Kdp were implicated as critical for K+ bindi ng from the analysis of mutants with reduced affinity for K+ (Buurman, E,, Rim, K,-T,, and Epstein, W, (1995) J, Biol Chem, 270, 6678-66851) K+ bindin g 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 ea ch of four binding clusters known to affect K+ binding. Amber mutations wer e constructed in a plasmid carrying the kdpFABC structural genes. Transferr ing these plasmids to 12 suppressor strains, each inserting a different ami no acid at amber codons, created 12 different substitutions at the mutated sites. This study delineates the four clusters and confirms that they are i mportant for K+ affinity but have little effect on the rate of transport. A t only 21 of the residues studied did at least three substitutions alter af finity 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 a ffinity, The effect of lysine is most likely a repulsive effect of this cat ionic residue on K+ and thus reflects the effective distance between a resi due and the site of binding or passage of K+ in Kdpk Once a crystallographi c structure of Kdp is available, this measure of effective distance will he lp identify the path of K+ as it moves through the KdpA subunit to cross th e membrane.