Abstract
Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. CBCRs bind bilins as chromophores and sense nearly the entire visible spectrum of light, but the regulation of the chromophorylation of CBCRs is unknown. Slr1393 from Synechocystis sp. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. The protein Slr2111 from Synechocystis sp. PCC 6803 includes a cystathionine β-synthase (CBS) domain pair of an as yet unknown function at its N terminus. CBS domains are often characterized as sensors of cellular energy status by binding nucleotides. In this work, we demonstrate that Slr2111 strongly interacts with Slr1393 in vivo and in vitro, which generates a complex in a 1:1 molar ratio. This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. Instead, the complex stability and thereby the chromophorylation of Slr1393 are regulated by the binding of nucleotides (ATP, ADP, AMP) to the CBS domains of Slr2111 with varying affinities. It is demonstrated that residues Asp-53 and Arg-97 of Slr2111 are involved in nucleotide binding. While ATP binds to Slr2111, the association between the two proteins gets weaker and chromophorylation of Slr1393 are enabled. In contrast, AMP binding to Slr2111 leads to a stronger association, thereby inhibiting the chromophorylation. It is concluded that Slr2111 acts as a sensor of the cellular energy status that regulates the chromophorylation of Slr1393 and thereby its function as a light-driven histidine kinase.
Highlights
Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors
The gene product is composed of three GAF domains, a PAS (Per, ARNT, and SIM protein domain) domain, and a histidine kinase (HK) domain
Our findings demonstrate a complex formation between the CBS domain-containing protein Slr2111 and the CBCR Slr1393 in vitro and in vivo, which blocks the entrance of the chromophore into the binding site of 1393g3 and thereby inhibits the assembly of Slr1393g3 with its PCB chromophore
Summary
Cyanobacteriochromes (CBCRs) are photochromic proteins in cyanobacteria that act as photosensors. PCC 6803 is a CBCR containing three consecutive GAF (cGMP phosphodiesterase, adenylyl cyclase, and FhlA protein) domains, of which only the third one (Slr1393g3) can be phycocyanobilin-chromophorylated. We demonstrate that Slr2111 strongly interacts with Slr1393 in vivo and in vitro, which generates a complex in a 1:1 molar ratio This tight interaction inhibits the chromophorylation of Slr1393g3, even if the chromophore is present. GAF3 (Slr1393g3, amino acids 423–599) is the sole domain that can bind PCB autocatalytically It yields a photochromic protein with absorption maxima at 649 nm (parental form) and 536 nm (photoproduct form) [10], which thereby light-regulates the activity of the C terminally located HK domain that binds ATP and autophosphorylates Slr1393. Some proteins consist of two or four tandem CBS domains, and others carry only one CBS domain; in plant-derived proteins, CBS domains occur more frequently in two CBS domains than in a single arrangement [14]
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