The Extension of the Fourth Transmembrane Helix of the Sensor Kinase KdpD ofEscherichia coliIs Involved in Sensing

Abstract

The KdpD sensor kinase and the KdpE response regulator control expression of the kdpFABC operon coding for the KdpFABC high-affinity K ؉ transport system of Escherichia coli. In search of a distinct part of the input domain of KdpD which is solely responsible for K ؉ sensing, sequences of kdpD encoding the transmembrane region and adjacent N-terminal and C-terminal extensions were subjected to random mutagenesis. Nine KdpD derivatives were identified that had lost tight regulation of kdpFABC expression. They all carried single amino acid replacements located in a region encompassing the fourth transmembrane helix and the adjacent arginine cluster of KdpD. All mutants exhibited high levels of kdpFABC expression regardless of the external K ؉ concentration. However, 3-to 14-fold induction was observed under extreme K ؉ -limiting conditions and in response to an osmotic upshift when sucrose was used as an osmolyte. These KdpD derivatives were characterized by a reduced phosphatase activity in comparison to the autokinase activity in vitro, which explains constitutive expression. Whereas for wild-type KdpD the autokinase activity and also, in turn, the phosphotransfer activity to KdpE were inhibited by increasing concentrations of K ؉ , both activities were unaffected in the KdpD derivatives. These data clearly show that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both K ؉ limitation and osmotic upshift, which may not be separated mechanistically.In bacteria K ϩ plays an important role in the maintenance of intracellular pH, cell turgor and control of cellular enzyme activities (for a review, see reference 5), and, together with glutamate, it is also known to positively regulate the expression of certain osmoresponsive genes (23, 32). The intracellular K ϩ level is maintained by a variety of K ϩ uptake and efflux systems. The KdpFABC complex (K ϩ -dependent ATPase), encoded by the kdpFABC operon (1), is an inducible, high-affinity K ϩ transporter synthesized by Escherichia coli as an emergency system to scavenge K ϩ when other transporters cannot keep up with the cell's requirement for K ϩ (4). Expression of the kdpFABC operon is regulated by the products of the adjacent kdpDE genes (31). The membrane-bound sensor kinase KdpD and the cytosolic response regulator KdpE comprise a typical prokaryotic two-component signal transduction system (43). KdpD (894 amino acids; 99 kDa) consists of a cytoplasmic N-terminal domain and a cytoplasmic C-terminal domain interconnected by four transmembrane segments (46). Upon stimulus perception, KdpD undergoes autophosphorylation at His-673, and subsequently the phosphoryl group is transferred to Asp-52 of the response regulator KdpE (42). KdpE binds in its phosphorylated and dimerized form with high affinity at a 23-bp sequence immediately upstream of the canonical Ϫ35 and Ϫ10 regions of the kdpFABC promoter, thereby triggering kdpFABC transcription (41). The large input domain of the sensor kinase KdpD (about 660 ...

The Universal Stress Protein UspC Scaffolds the KdpD/KdpE Signaling Cascade of Escherichia coli under Salt Stress

“…14 A putative K + -binding site is suspected within the cytoplasmic extension of transmembrane helix IV. 15,16 The effect of K + on KdpD activation was corroborated by the observation that transcription of kdpFABC is also induced when cells are grown in the presence of Cs + , a known inhibitor of K + uptake systems. 11 (iii) Studies with KdpD inserted in rightside-out membrane vesicles, in which the cytoplasmic domains are unidirectionally exposed to the lumen of the vesicles, indicated that an increase of the intraluminal ionic strength stimulates the kinase activity.…”

A lipid-mediated conformational switch modulates the thermosensing activity of DesK

“…Interestingly, a cluster of positive and negative residues located at the water-lipid interface is also present in the tandempore K channel (TREK-1), the potassium sensor KdpD, the transient receptor potential proteins (TRPs), the OpuA transporter, and the mechanosensitive MscL channels ( Table 2). For some of them, it has been suggested that the charged residues could interact with the negative surface of the membrane, modulating activity (15)(16)(17)(18). We propose that highly charged residue clusters involved in signaling proteins and channels (Table 2) are able to adopt alternative states (unbound or bound to the membrane) in response to changes in mechanical properties of the lipid membrane, allowing this region to behave as a molecular switch for downstream protein activity.…”

Reduction of Turgor Is Not the Stimulus for the Sensor Kinase KdpD ofEscherichia coli

“…Sugiura et al (37) suggested mechanistically different sensing mechanisms for potassium-limiting conditions and osmotic stress. However, Zimmann et al (44) showed that the extension of the fourth transmembrane helix encompassing the arginine cluster is mainly involved in sensing both stimuli, which may not be separable. Furthermore, Heermann et al (14) proposed an additional, more-complex regulatory network for kdpFABC expression.…”