The hydrophilic N-terminal domain complements the membrane-anchored C-terminal domain of the sensor kinase KdpD of Escherichia coli

The putative turgor sensor KdpD is characterized by a large, N-terminal dom ain of about 400 amino acids, which is not found in any other known sensor kinase. Comparison of 12 KdpD sequences from various microorganisms reveals that this part of the kinase is highly conserved and includes two motifs ( Walker A and Walker B) that are very similar to the classical ATP-binding s ites of ATP-requiring enzymes. By means of photoaffinity labeling with 8-az ido-[alpha-P-32]ATP, direct evidence was obtained for the existence of an A TP-binding site located in the N-terminal domain of KdpD. The N-terminal do main, KdpD/1-395, was overproduced and purified. Although predicted to be h ydrophilic, it was found to be membrane-associated and could be solubilized either by treatment with buffer of low ionic strength or detergent. The me mbrane-associated form, but not the solubilized one, retained the ability t o bind 8-azido-[alpha-P-32]ATP. Previously, it was shown that the phosphata se activity of a truncated KdpD, KdpD/Delta 12-395, is deregulated in vitro (Jung, K., and Altendorf, K. (1998) J. Biol. Chem. 273, 17406-17410). Here , we demonstrated that this effect was reversed in vesicles containing both the truncated KdpD and the N-terminal domain. Furthermore, coexpression of kdpD/Delta 12-395 and kdpD/1-395 restored signal transduction in vivo. The se results highlight the importance of the N-terminal domain for the functi on of KdpD and provide evidence for an interaction of this domain and the t ransmitter domain of the sensor kinase.