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 membr ane-bound sensor kinase KdpD and the cytoplasmic response regulator KdpE. H ere, we examine the oligomeric state of KdpD. KdpD-His(673) --> Gln and Kdp D-Asn(788) --> Asp are kinase inactive. When the corresponding genes are co expressed, the resulting KdpD protein regains kinase activity in vitro, sug gesting 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-(Delta 128-391) leads to stable heterooligomers that can bind to Ni-NTA agarose and that are coelute d. Purified and solubilized KdpD-6His has been electrophoresed in blue nati ve polyacrylamide gels (BN-PAGE), and unphosphorylated and phosphorylated K dpD 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 KdpB-6His and KdpD-6His similar to P-32 by gel fil tration reveals a value of 245 kDa for both forms of the protein. The Stoke s radius is determined to be 5.4 nm. sucrose gradient sedimentation analysi s of KdpD-6His results in a molecular mass of 289 kDa. The calculated molec ular mass of a KdpD-6His monomer is 99.6 kDa. Considering the detergent bou nd to KdpD the obtained data reveal that KdpD is a homodimer and there is n o change in the oligomeric state upon activation. Crosslinking experiments with single Cys KdpD molecules indicate that there is a close contact betwe en the monomers in the transmitter as well as in transmembrane domain 1. BN -PAGE of solubilized and purified KdpD-6His devoid of Cys residues demonstr ates that Cys residues do not contribute to the stabilization of the dimer. (C) 1998 Elsevier Science B.V. All rights reserved.