Individual substitutions of clustered arginine residues of the sensor kinase KdpD of Escherichia coli modulate the ratio of kinase to phosphatase activity.

Escherichia coli responds to K+ limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating Kdp-ATPase. KdpD, the sensor kinase of this system, is a bifunctional enzyme catalyzing the autophosphorylation by ATP and the dephosphorylation of the corresponding response regulator KdpE. Here we demonstrate that individual replacements of clustered arginine residues located close to transmembrane domain TM4 modulate the ratio of kinase to phosphatase activity. Thus KdpD-Arg511 --> Gln is characterized by an increase in the kinase activity and a loss of the phosphatase activity. However, when Arg at position 511 is replaced with Lys, activities of the corresponding protein are comparable with wild-type KdpD. In contrast, replacement of arginine residues at positions 503, 506, or 508 with glutamine or lysine causes a decrease of the kinase and an increase of the phosphatase activities. Changes of the activities of these KdpD proteins correspond with alterations in kdpFABC expression. Thus KdpD-Arg511 --> Gln causes constitutive expression of kdpFABC. KdpD proteins with Arg replacements at positions 503, 506, or 508 are unable to respond to osmolarity, whereas the sensing of K+ limitation is not influenced. Simultaneous replacement of arginine residues 508 and 511 or 506, 508, and 511 with glutamine leads to a decrease of the phosphatase activity. However, kdpFABC expression is dependent on K+ and osmolarity. Finally, when Arg513 is replaced with glutamine the amount of KdpD detected in the membrane is drastically reduced. These results imply that there is an equilibrium between the kinase and phosphatase activities of KdpD, which can be shifted by the replacement of one arginine residue. An electrostatic switch mechanism within the protein is proposed through which the ratio of kinase to phosphatase is regulated. Finally, these results lend support to the notion that KdpD can be activated by two distinct stimuli, K+ limitation and osmolarity.