Biochemical Properties of CikA, an Unusual Phytochrome-like Histidine Protein Kinase That Resets the Circadian Clock in Synechococcus elongatus PCC 7942

Abstract

We recently described the cikA (circadian input kinase A) gene, whose product supplies environmental information to the circadian oscillator in the cyanobacterium Synechococcus elongatus PCC 7942. CikA possesses three distinct domains: a GAF, a histidine protein kinase (HPK), and a receiver domain similar to those of the response regulator family. To determine how CikA functions in providing circadian input, we constructed modified alleles to tag and truncate the protein, allowing analysis of each domain individually. CikA covalently bound bilin chromophores in vitro, even though it lacks the expected ligand residues, and the GAF domain influenced but did not entirely account for this function. Full-length CikA and truncated variants that carry the HPK domain showed autophosphorylation activity. Deletion of the GAF domain or the N-terminal region adjacent to GAF dramatically reduced autophosphorylation, whereas elimination of the receiver domain increased activity 10-fold. Assays to test phosphorelay from the HPK to the cryptic receiver domain, which lacks the conserved aspartyl residue that serves as a phosphoryl acceptor in response regulators, were negative. We propose that the cryptic receiver is a regulatory domain that interacts with an unknown protein partner to modulate the autokinase activity of CikA but does not work as bona fide receiver domain in a phosphorelay.

The pseudo‐receiver domain of CikA regulates the cyanobacterial circadian input pathway

“…Because two-component signal transduction systems are common in bacteria (Stock et al ., 2000), and CikA is a canonical HPK, it likely has a cognate response regulator (RR), a hypothetical partner termed CikR. The PsR domain of CikA cannot be phosphorylated by CikA HPK activity, consistent with absence from the PsR of the conserved aspartic acid present in bona fide RR proteins that receives a phosphoryl group from an HPK (Mutsuda et al ., 2003); thus, the PsR domain is not CikR. PsR domains may function like the receivers of RRs in regulating an adjacent domain, but use protein-protein interactions rather than phosphorylation to effect conformational change (O'Hara et al ., 1999;Williams et al ., 2002).…”

“…All constructs were integrated at a neutral site (NS I) in the S. elongatus chromosome, and driven by a P trc promoter, which is inducible by isopropyl-β -D -thiogalactopyranoside (IPTG). The P trc promoter exhibits low-level basal expression in the cyanobacterium (Mutsuda et al ., 2003), such that we could test both complementation in uninduced conditions, and an overexpression phenotype in the presence of inducer, for all constructs (Table 1). Circadian phenotypes were monitored by the oscillation in bioluminescence from a kaiB::luxAB reporter strain, which tracks circadian expression from the kaiBC promoter.…”

“…CikA variants have decreased kinase activity in the absence of the featureless 183-aa N-terminal segment, and nondetectable kinase activity without the GAF or HPK, or with a point mutation in the catalytic histidine of the HPK. Conversely, a 10-fold increased kinase activity when the PsR domain is removed reveals PsR as a negative regulator of kinase activity (Mutsuda et al ., 2003). Because two-component signal transduction systems are common in bacteria (Stock et al ., 2000), and CikA is a canonical HPK, it likely has a cognate response regulator (RR), a hypothetical partner termed CikR.…”

“…However, the GAF of CikA lacks a conserved cysteine or histidine to serve as a bilin-binding site. Moreover, CikA purified from the cyanobacterium is negative in a Zinc-fluorescence assay that tests for the presence of an attached bilin, leaving the nature of a potential in vivo cofactor still mysterious (Mutsuda et al ., 2003). In vitro autophosphorylation assays established that CikA is a bona fide kinase and demonstrated that the presence of other domains regulates its kinase activity (Mutsuda et al ., 2003).…”

“…Moreover, CikA purified from the cyanobacterium is negative in a Zinc-fluorescence assay that tests for the presence of an attached bilin, leaving the nature of a potential in vivo cofactor still mysterious (Mutsuda et al ., 2003). In vitro autophosphorylation assays established that CikA is a bona fide kinase and demonstrated that the presence of other domains regulates its kinase activity (Mutsuda et al ., 2003). CikA variants have decreased kinase activity in the absence of the featureless 183-aa N-terminal segment, and nondetectable kinase activity without the GAF or HPK, or with a point mutation in the catalytic histidine of the HPK.…”