Purification, reconstitution, and characterization of KdpD, the turgor sensor of Escherichia coli

Abstract

In response to K+ availability or medium osmolality, the sensor kinase KdpD and the response regulator KdpE control the expression of the kdpFABC operon, coding for the high affinity K+-translocating Kdp ATPase of Escherichia coli. The stimulus for KdpD to undergo autophosphorylation is believed to be a change in turgor or some effect thereof, reflecting the role of K+ as an important cytoplasmic osmotic solute, The membrane-bound sensor kinase KdpD was overproduced as a fusion protein containing six contiguous histidine residues two amino acids before the C terminus. This KdpD-His(6), protein was functional in vitro and in vivo, KdpD-His(6) was purified from everted membrane vesicles by solubilization with the zwitterionic detergent lauryldimethylamine oxide followed by nickel chelate chromatography and ion exchange chromatography to >99% homogeneity. The solubilized protein was not active with respect to autophosphorylation, but retained the ability to bind a-azido-ATP. KdpD-His(6), was reconstituted into proteoliposomes in a unidirectional inside-out orientation as revealed by ATP accessibility and protease susceptibility. Purified and reconstituted KdpD-His(6), exhibited autokinase activity, and the phosphoryl group could be transferred to KdpE, Furthermore, KdpD-His(6), was found to be the only protein that mediates dephosphorylation of KdpE similar to P.