Conserved mechanism for sensor phosphatase control of two-component signaling revealed in the nitrate sensor NarX

Abstract

Two-component signal transduction mediates a wide range of phenotypes in microbes and plants. The sensor transmitter module controls the phosphorylation state of the cognate-responseregulator receiver domain. Whereas the two-component autokinase and phosphotransfer reactions are well-understood, the mechanism by which sensors accelerate the rate of phosphoresponse regulator dephosphorylation, termed "transmitter phosphatase activity," is unknown. We identified a conserved DxxxQ motif adjacent to the phospho-accepting His residue in the HisKA_3 subfamily of two-component sensors. We used site-specific mutagenesis to make substitutions for these conserved Gln and Asp residues in the nitrate-responsive NarX sensor and analyzed function both in vivo and in vitro. Results show that the Gln residue is critical for transmitter phosphatase activity, but is not essential for autokinase or phosphotransfer activities. The documented role of an amide moiety in phosphoryl group hydrolysis suggests an analogous catalytic function for this Gln residue in HisKA_3 members. Results also indicate that the Asp residue is important for both autokinase and transmitter phosphatase activities. Furthermore, we noted that sensors of the HisKA subfamily exhibit an analogous E/DxxT/N motif, the conserved Thr residue of which is critical for transmitter phosphatase activity of the EnvZ sensor. Thus, twocomponent sensors likely use similar mechanisms for receiver domain dephosphorylation.histidine kinase | DHp domain | DesK

Two-component signal transduction systems in oral bacteria

“…The E/D residue is required for kinase and N/T for phosphatase activities, respectively [64,65]. Experimentally, it was demonstrated that the recombinant cytoplasmic fragment of HaeS uses ATP as the phosphodonor to autophosphorylate the conserved histidine 226 residue, while a recombinant cytoplasmic fragment of HaeS carrying the H226A point mutation was not phosphorylated.…”

Sensor–response regulator interactions in a cross-regulated signal transduction network

“…This highlights the substantial differences in detail for reaction mechanisms employed by the two transmitter sequence families (Huynh et al, 2010).…”

“…Yang & Inouye (1991) applied this logic to the sensor transmitter domain, demonstrating intragenic complementation between alleles that inactivate either the DHp or the CA domain. For the narX and narQ genes, we used missense substitutions that remove the DHp domain phospho-accepting His residue (residues His-399 and His-370 in NarX and NarQ, respectively) together with substitutions that abolish CA domain function (residues His-513 and His-484, respectively) (Huynh et al, 2010). Allele pairs were expressed from the compatible plasmids pHG165 and pSU18 (Table 1), and sensor function was examined in a W(narG-lacZ) DnarX DnarQ double null strain background.…”

Type IV pilins regulate their own expression via direct intramembrane interactions with the sensor kinase PilS

“…Recently, a conserved E/DxxN/T phosphatase motif adjacent to the phosphorylated His was identified in the HisKA family of SKs to which PilS belongs, indicating that many SKs can have dual kinase and phosphatase activities to fine tune regulation (23). PilS has a canonical ExxN motif at position 320-323, beside the H319 phosphorylation site.…”