K. Jung et al., EFFECT OF CYSTEINE REPLACEMENTS ON THE PROPERTIES OF THE TURGOR SENSOR KDPD OF ESCHERICHIA-COLI, Biochimica et biophysica acta. Biomembranes, 1372(2), 1998, pp. 311-322
Escherichia coli responds rapidly to K+-limitation or high osmolarity
by induction of the kdpFABC operon coding for the high affinity K+-tra
nslocating Kdp-ATPase. This process is controlled by the membrane-boun
d histidine kinase KdpD and the response regulator KdpE. Here, it is d
emonstrated that replacements of the native Cys residues at positions
409, 852, and 874 influence distinct activities of KdpD, whereas repla
cements of Cys residues at positions 32, 256, and 402 have no effect.
Replacements of Cys(409) in KdpD reveal that transmembrane domain I is
important for perception and/or propagation of the stimulus. When Cys
(409) is replaced with Ala, kdpFABC expression becomes constitutive re
gardless of the external stimuli. In contrast, when Cys(409) is replac
ed with Val or Tyr, induction of kdpFABC expression in response to dif
ferent stimuli is drastically reduced. KdpD with Ser at position 309 s
upports levels of kdpFABC expression comparable to those seen in wildt
ype. Since neither the kinase nor phosphatase activity of these protei
ns is affected, it is proposed that different amino acid side-chains a
t position 409 alter the switch between the inactive and active forms
of the kinase. When Cys(852) or Cys(874) is replaced with Ala or Ser,
kinase activity is reduced to 10% of the wild-type level. However, kin
etic studies reveal that the apparent ATP binding affinity is not affe
cted. Surprisingly, introduction of Cys(852) and Cys(874) into a KdpD
protein devoid of Cys residues leads to full recovery of the kinase ac
tivity. Labeling studies support the idea that a disulfide bridge form
s between these two residues. (C) 1998 Elsevier Science B.V. All right
s reserved.