Truncation of Amino Acids 12–128 Causes Deregulation of the Phosphatase Activity of the Sensor Kinase KdpD of Escherichia coli

Abstract

The kdpFABC operon, which encodes the structural genes for the high affinity K ؉ transport complex KdpFABC, is regulated by the sensor kinase KdpD and the response regulator KdpE. KdpD is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE. Here, we demonstrate that the phosphatase activity of KdpD is dependent on ATP, whereas GTP, ITP, CTP, ADP, and GDP have no effect. The phosphatase activity requires only ATP binding, because nonhydrolyzable analogs (adenosine-5 -[␥-thio]triphosphate and adenosine-5 -[␤,␥-imido]triphosphate) work as well. However, KdpD proteins missing amino acids 12-128 are characterized by a phosphatase activity that is independent of ATP. These proteins are still able to respond to K ؉ starvation, but an increase in osmolarity is no longer sensed. Comparison of different KdpD sequences reveals a conserved motif in this amino acid region that is very similar to a classical ATP-binding site (Walker A motif). Replacement of the conserved Gly 37 , Lys 38 , and Thr 39 residues in the consensus ATP-binding sequence results in a KdpD protein that causes a kdp-FABC expression pattern comparable with that seen with KdpD proteins missing amino acids 12-128. However, in vitro phosphatase activity is comparable with that of wild-type KdpD. These results suggest that amino acids 12-128 of KdpD are important for its activity and that an additional ATP-binding site in the Nterminal region seems to be involved in modulation of the phosphatase activity.

The Hydrophilic N-terminal Domain Complements the Membrane-anchored C-terminal Domain of the Sensor Kinase KdpD ofEscherichia coli

“…This presumption is supported by the differentiated response of KdpD proteins with individual substitutions of Arg residues toward K ϩ limitation and osmotic upshock (9). Compared with other known sensor kinases, KdpD is the only protein that has a large, hydrophilic N-terminal domain of about 400 amino acids in length (17,18) (Fig. 1) that this domain is more conserved then other regions of KdpD.…”

“…Truncated KdpD proteins lacking different parts of the N-terminal domain, including amino acids 12-128 are characterized by phosphatase activities independent of the presence of ATP. This finding suggests that an ATP-binding site has a regulatory role within this domain (18).…”

“…(ii) The four transmembrane domains seem to be important for the correct positioning of N-and C-terminal domains to each other as well as for perception and/or signal transduction (7,17,22). (iii) Amino acids 12-128, including a slightly modified Walker A site, seem to be important for the regulation of the phosphatase activity of KdpD (18). Here, we describe the overexpression and purification of the N-terminal domain of KdpD as well as its functional importance.…”

“…1) that this domain is more conserved then other regions of KdpD. This N-terminal domain includes two sequence motifs, which are very similar to those of a typical ATP-binding site (Walker A and B motifs) (18) (Fig. 2).…”

“…Plasmid pWP6-92 carries an in-frame deletion of 852 base pairs, encoding KdpD/⌬12-395 (17). Plasmid pPV5-1/G37A, K38A, T39C 2 encodes KdpD/G37A, K38A, T39C (18). In plasmid pBD, kdpD was cloned into pBAD18 (23), in which expression of kdpD is under control of the arabinose promoter (22).…”