KdpD consists of a characteristic C-terminal transmitter domain, which is fused via a small linker region to the large N-terminal input domain. Several regions of the input domain have been identified as important for stimulus perception and integration. The four Selleck GSK458 transmembrane domains (TM1-TM4) anchor the sensor kinase in the cytoplasmic membrane and separate the two large cytoplasmic

domains from each other [7, 8]. The transmembrane helices TM2 and TM3 function as a type of clip and are responsible for the correct positioning of the large cytoplasmic domains relative to each other [8]. We have previously shown a direct interaction between these KdpD cytoplasmic domains [9]. The α-helix of TM4 extends from the membrane into the cytoplasm and encompasses a cluster of positively charged amino acids (R503-R511) that are mainly involved in stimulus perception, and has therefore been Ralimetinib proposed as a K+ binding site by Altendorf and coworkers [10, 11]. This hypothesis is in accord with the finding that amino acid replacements resulting in K+-independent kdpFABC expression are located within TM4 and the adjacent region [11–13]. It was previously shown that the cluster of positively charged Vactosertib cost amino acids is important for modulation

of the kinase and phosphatase activity, because individual replacements of these amino acids resulted in KdpD derivatives with either enhanced kinase and reduced phosphatase activity, or enhanced phosphatase and reduced kinase activity [10]. Furthermore, a KdpD derivative lacking until the cytoplasmic N-terminal region and the first two transmembrane domains of KdpD were able to respond to K+ limitation, which supports the assumption that the K+ binding site is located within this region [14]. The role of the KdpD N-terminal input domain large cytoplasmic region (M1-W395, Fig. 1) for sensing and signal transduction has been a mystery for a long time. Altendorf and coworkers

found that truncations within the N-terminal domain resulted in functional KdpD protein in vitro [15]. Later, a sequence motif was identified within this domain that is very similar to the classical “”Walker A”" motif [16]. Truncations that encompass this motif (R12-D228, R12-W395) result in deregulated phosphatase activity [16]. Since ATP-binding within this region is known to be involved in modulation of the phosphatase activity, ATP may function as an intracellular stimulus that is sensed by KdpD under osmotic stress [9, 16]. This is in accord with the finding that the intracellular ATP concentration increases significantly upon an osmotic upshift [17]. A truncated version of KdpD comprising only the N-terminal cytoplasmic domain (KdpD/1-395) caused constitutive expression of kdpFABC in vivo, revealing a stabilizing function of the N-terminal domain of KdpD in complex with phosphorylated KdpE and the corresponding DNA binding site [8].