ATP binding to the KTN/RCK subunit KtrA from the K+- uptake system KtrAB of Vibrio alginolyticus - Its role in the formation of the KtrAB complex and its requirement in vivo

Abstract

Subunit KtrA of the bacterial Na+-dependent K+-translocating KtrAB systems belongs to theKTN/RCKfamily 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 (His(10)-VaKtrA) and isolated by affinity chromatography. VaKtrA contains a G-rich, ADP-moiety binding beta-alpha-beta-fold (''Rossman fold''). Photocross-linking and flow dialysis were used to determine the binding of [P-32] ATP and [P-32] NAD(+) to His(10)-VaKtrA. Binding of other nucleotides was estimated from the competition by these compounds of the binding of the P-32-labeled nucleotides to the protein. [gamma-P-32] ATP bound with high affinity to His(10)-VaKtrA (K-D of 9 mu M). All other nucleotides tested exhibited K-D (K-i) values of 30 mu M 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-His(6), as detected both in an overlay experiment and in an experiment in which VaKtrA was added to VaKtrB-His(6) 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.