Effect of cysteine replacements on the properties of the turgor sensor KdpD of Escherichia coli.
Escherichia coli responds rapidly to K+-limitation or high osmolarity by induction of the kdpFABC operon coding for the high affinity K+-translocating Kdp-ATPase. This process is controlled by the membrane-bound histidine kinase KdpD and the response regulator KdpE. Here, it is demonstrated that replacements of the native Cys residues at positions 409, 852, and 874 influence distinct activities of KdpD, whereas replacements of Cys residues at positions 32, 256, and 402 have no effect. Replacements of Cys409 in KdpD reveal that transmembrane domain I is important for perception and/or propagation of the stimulus. When Cys409 is replaced with Ala, kdpFABC expression becomes constitutive regardless of the external stimuli. In contrast, when Cys409 is replaced with Val or Tyr, induction of kdpFABC expression in response to different stimuli is drastically reduced. KdpD with Ser at position 409 supports levels of kdpFABC expression comparable to those seen in wild-type. Since neither the kinase nor phosphatase activity of these proteins is affected, it is proposed that different amino acid side-chains at position 409 alter the switch between the inactive and active forms of the kinase. When Cys852 or Cys874 is replaced with Ala or Ser, kinase activity is reduced to 10% of the wild-type level. However, kinetic studies reveal that the apparent ATP binding affinity is not affected. Surprisingly, introduction of Cys852 and Cys874 into a KdpD protein devoid of Cys residues leads to full recovery of the kinase activity. Labeling studies support the idea that a disulfide bridge forms between these two residues.
Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Universität Osnabrück, D-49069 Osnabrück, Germany. jung_k@sfbbiol.biologie.uni-osnabrueck.de
K
K
Jung
R
R
Heermann
M
M
Meyer
K
K
Altendorf
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine; D004220:Disulfides / Q000032:analysis / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry*; D029968:Escherichia coli Proteins; D007700:Kinetics; D008081:Liposomes; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; D008081:Liposomes; C489580:KdpD protein, E coli; D011494:Protein Kinases; C075616:kdpD protein, Bacteria; D003545:Cysteine