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

Steve Dorus, Haruo Mimura, Wolfgang Epstein

Research output: Contribution to journalArticle

14 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)9590-9598
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number13
DOIs
StatePublished - Mar 30 2001
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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