Genetic and Biochemical Dissection of a HisKA Domain Identifies Residues Required Exclusively for Kinase and Phosphatase Activities

Abstract

Two-component signal transduction systems, composed of histidine kinases (HK) and response regulators (RR), allow bacteria to respond to diverse environmental stimuli. The HK can control both phosphorylation and subsequent dephosphorylation of its cognate RR. The majority of HKs utilize the HisKA subfamily of dimerization and histidine phosphotransfer (DHp) domains, which contain the phospho-accepting histidine and directly contact the RR. Extensive genetics, biochemistry, and structural biology on several prototypical TCS systems including NtrB-NtrC and EnvZ-OmpR have provided a solid basis for understanding the function of HK–RR signaling. Recently, work on NarX, a HisKA_3 subfamily protein, indicated that two residues in the highly conserved region of the DHp domain are responsible for phosphatase activity. In this study we have carried out both genetic and biochemical analyses on Myxococcus xanthus CrdS, a member of the HisKA subfamily of bacterial HKs. CrdS is required for the regulation of spore formation in response to environmental stress. Following alanine-scanning mutagenesis of the α1 helix of the DHp domain of CrdS, we determined the role for each mutant protein for both kinase and phosphatase activity. Our results indicate that the conserved acidic residue (E372) immediately adjacent to the site of autophosphorylation (H371) is specifically required for kinase activity but not for phosphatase activity. Conversely, we found that the conserved Thr/Asn residue (N375) was required for phosphatase activity but not for kinase activity. We extended our biochemical analyses to two CrdS homologs from M. xanthus, HK1190 and HK4262, as well as Thermotoga maritima HK853. The results were similar for each HisKA family protein where the conserved acidic residue is required for kinase activity while the conserved Thr/Asn residue is required for phosphatase activity. These data are consistent with conserved mechanisms for kinase and phosphatase activities in the broadly occurring HisKA family of sensor kinases in bacteria.

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.…”

Genetic analysis of signal integration by the Sinorhizobium meliloti sensor kinase FeuQ

“…We were unable to perform the converse experiment by selectively eliminating phosphatase activity. In some sensor kinases it has been shown that within a conserved E/DxxT motif of the DHp domain, the Thr residue is critical for phosphatase activity (Huynh & Stewart, 2011;Willett & Kirby, 2012). FeuQ shares this conserved motif, but mutations to the conserved Thr did not give a phenotype consistent with eliminated phosphatase activity (data not shown).…”

“…We performed random mutagenesis of feuQ searching for 'FeuN-insensitive' (constitutively activating) and 'FeuN-mimicking' (constitutively inhibiting) alleles that might reveal domains of the FeuQ protein that are important for potential FeuN interaction and toggling between kinase and phosphatase activities. We expected to find mutations leading to each phenotype in the HAMP, DHp and CA domains, as these regions are known to be involved in SK signal integration (Atkinson & Ninfa, 1993;Hsing et al, 1998;Willett & Kirby, 2012). We suspected that mutations might also correspond to the FeuQ periplasmic domain, where FeuQ and FeuN are most likely to directly interact.…”

Chemosensory Regulation of a HEAT-Repeat Protein Couples Aggregation and Sporulation in Myxococcus xanthus

“…Proteins were radiolabeled with acetyl phosphate ([ 32 P]AcP) as described previously (10,24). Briefly, [ 32 P]AcP was generated using E. coli acetate kinase (Sigma).…”