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

Abstract

The Kdp-ATPase of Escherichia coli is a four-subunit P-type ATPase that accumulates K ؉ with high affinity and specificity. ؉ 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.

Amino Acid Substitutions in Putative Selectivity Filter Regions III and IV in KdpA Alter Ion Selectivity of the KdpFABC Complex from Escherichia coli

“…In the other cases, only KdpC, containing the histidine motif, was eluted from Ni-NTA. This observation emphasizes the possibility that the stability of the complex is tightly coupled with correctly folded subunits and might be one explanation for the finding in previous studies that these residues could not be changed (4). The substitutions at S471 did not affect K ϩ -and Rb ϩ -stimulated ATPase activity, but ammonium was now effective.…”

“…1A). However, some of the mutant complexes (the G345A, G345S, G470S, and S471G mutants) did show NH 4 ϩ -stimulated ATPase activity, ruling out the pos- sibility that the histidine motif, which was not present in the complex used in the previous report, might have abolished the ammonium effect. The purification procedure described here leads to a protein preparation that is much more homogeneous than the previous one.…”

“…Sequence alignment (6, 7) and mutagenesis studies (3,4,18) lend support to the notion that KdpA is the K ϩ -binding and -translocating subunit of the KdpFABC complex. Beside the general agreement that KdpA has selectivity filter regions similar to those found in KcsA (5), the number and localization of the filter regions in KdpA remained unclear.…”

“…Furthermore, in the case of Arabidopsis thaliana HKT, the N-terminal glycine residue within the first selectivity filter is replaced by serine, a change that is perhaps correlated with the shift in selectivity from K ϩ to Na ϩ (17). The second putative filter region of KdpA (residues G232, G233, and G234) has been well characterized (3,4,14,18). In particular, residue G232 was of fundamental importance for ion selectivity (14,18), whereas residues G233 and G234 had only a minor effect (18).…”

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

“…Peak intensities were extracted using SPARKY. Relaxation rates were obtained from fitting the intensities to an exponential decay using CURVEFIT 7 and the perlscript SPARKY2RATE. 8 A minimum uncertainty of 2% for all rates was assumed.…”