ATP Binding to the KTN/RCK Subunit KtrA from the K+-uptake System KtrAB of Vibrio alginolyticus

Subunit KtrA of the bacterial Na+-dependent K+-translocating KtrAB systems belongs to the KTN/RCK family of regulatory proteins and protein domains. They are located at the cytoplasmic side of the cell membrane. By binding ligands they regulate the activity of a number of K+ transporters and K+ channels. To investigate the function of KtrA from the bacterium Vibrio alginolyticus (VaKtrA), the protein was overproduced in His-tagged form (His10-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding β-α-β-fold (“Rossman fold”). Photocross-linking and flow dialysis were used to determine the binding of [32P]ATP and [32P]NAD+ to His10-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the 32P-labeled nucleotides to the protein. [γ-32P]ATP bound with high affinity to His10-VaKtrA (KD of 9 μm). All other nucleotides tested exhibited KD (Ki) values of 30 μm or higher. Limited proteolysis with trypsin showed that ATP was the only nucleotide that changed the conformation of VaKtrA. ATP specifically promoted complex formation of VaKtrA with the His-tagged form of its K+-translocating partner, VaKtrB-His6, as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His6 bound to Ni2+-agarose. In intact cells of Escherichia coli both a high of membrane potential and a high cytoplasmic ATP concentration were required for VaKtrAB activity. C-terminal deletions in VaKtrA showed that for in vivo activity at least 169 N-terminal amino acid residues of its total of 220 are required and that its 40 C-terminal residues are dispensable.