The turgor sensor KdpD of Escherichia coli is a homodimer.

Escherichia coli responds to K+-limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating KdpFABC complex. Expression of the corresponding operon is controlled by the membrane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE. Here, we examine the oligomeric state of KdpD. KdpD-His673-->Gln and KdpD-Asn788-->Asp are kinase inactive. When the corresponding genes are coexpressed, the resulting KdpD protein regains kinase activity in vitro, suggesting that the functional state of KdpD is at least a dimer and that the kinase reaction is a result of a trans-phosphorylation between two monomers. Furthermore, coexpression of kdpD-6His and kdpD-(Delta128-391) leads to stable heterooligomers that can bind to Ni-NTA agarose and that are coeluted. Purified and solubilized KdpD-6His has been electrophoresed in blue native polyacrylamide gels (BN-PAGE), and unphosphorylated and phosphorylated KdpD resulted in the same band pattern suggesting that the oligomeric state of KdpD does not change upon phosphorylation. In addition, determination of the molecular masses of KdpD-6His and KdpD-6His approximately 32P by gel filtration reveals a value of 245 kDa for both forms of the protein. The Stokes radius is determined to be 5.4 nm. Sucrose gradient sedimentation analysis of KdpD-6His results in a molecular mass of 289 kDa. The calculated molecular mass of a KdpD-6His monomer is 99.6 kDa. Considering the detergent bound to KdpD the obtained data reveal that KdpD is a homodimer and there is no change in the oligomeric state upon activation. Crosslinking experiments with single Cys KdpD molecules indicate that there is a close contact between the monomers in the transmitter as well as in transmembrane domain 1. BN-PAGE of solubilized and purified KdpD-6His devoid of Cys residues demonstrates that Cys residues do not contribute to the stabilization of the dimer.