Chromophorylation Of Cyanobacteriochrome Slr1393 From Synechocystis Sp.PCC 6803 Is Regulated By Protein Slr2111 Through Allosteric Interaction

Cyanobacteriochromes?CBCR?are photochromic proteins in cyanobacteria that act as photosensors.CBCR bind bilins as chromophores and sense nearly the entire visible spectrum of light.However,as the chromophorylation of CBCR and similar phytochromes is an autocatalytic reaction,and the regulation of the chromophorylation of CBCR is unknown.Only the third GAF domain?Slr1393g3?of CBCR Slr1393 from Synechocystis sp.PCC 6803?Synechocystis?can be phycocyanobilin-chromophorylated,the complex forms a red and green reversible pigment protein.The light-regulates the activity of the C-terminally located HK domain,which binds ATP and autophosphorylates Slr1393,then interact with the response regulator of two-components regulatory system and influence the transcription level of related genes.Slr2111 from the unicellular cyanobacterium Synechocystis was annotated as a hypothetical protein with unknown function,contains two domains:a tandem arrangement of two conservative CBS?cystathionine beta-synthase?domains and a segment assigned to the aspartate aminotransferase?AAT?.CBS domain are small protein modules?typically 60 residues in lengh?,packed a?1-?1-?2-?3-?2 topology,either as strand-alone proteins or fused to thousands of cytosolic or transmembrance protein domains.The CBS domain is an evolutionarily conserved protein domain that is present in the proteome of prokaryotes and eukaryotes.The CBS domains occur in tandem pairs,forming a so-called CBS pair or Bateman module.CBS domains are fused to other functional domain?s?,which regulate their probable functions through binding of ligands.CBS domain could bind ATP,ADP,AMP and SAM,which lead the conformation change of the protein,CBS domains regulate cellular functions in response to changes in energy status.Data from a large-scale protein-protein interaction screening for Synechocystis indicate that Slr1393 might interact with Slr2111.In this work,the interaction between Slr2111 and Slr1393 was analyzed in detail.Employment of bacterial two-hybrid confirmed the strong interaction between Slr2111and Slr1393g3.Through gel filtration chromatography and pull-down assay confirmed the complex formation between the two independent proteins Slr1393 and Slr2111 in a 1:1molar ratio.The Zn²⁺induced fluorescence of the sample of pull-down and co-immunoprecipitation indicates that Slr2111 only interact with apo-form of Slr1393.The CBS domain and AAT domain is necessary for the interaction of Slr2111 and Slr1393,and Slr2111 only interact with Slr1393g3 among the domains of Slr1393.We quantified the affinity of Slr2111 for adenosine nucleotides by measuring its intrinsic fluorescence.Using the modified Stern–Volmer plot,significant quenching of the fluorescence intensity of Slr2111 was recorded upon addition of ATP,ADP and AMP,yielding the dissociation constants:AMP shown the highest affinity with Slr2111,the affinity of ATP is 2-to 3-fold lower.The circular dichroism spectra of Slr2111 upon addition of adenosine nucleotides revealed significant changes of protein secondary structures,indicaring that AMP-binding have variously strong allosteric effects on Slr2111.The adenosyl binding site was confirmed by mutation of amino acid:as the 53th amino aspartic acid and the 97th amino arginine of Slr2111 were mutated,the mutations have significantly reduced the affinity of binding nucleotide,and the circular dichroism spectra of Slr2111 mutants upon addition of adenosine nucleotides indicates that allosteric effects were slightly changed.The interaction of Slr1393g3 and Slr2111 blocks the entrance of the chromophore into the binding site of 1393g3 and thereby inhibits the assembly of Slr1393g3 with its PCB chromophore.Through enzyme kinetics Slr2111 has a competitive inhibition on Slr1393g3 binding PCB.Slr2111/AMP and Slr2111/ATP also show competitive inhibition on chromophorylation of Slr1393g3 at low concentrations.The fact that the K_i value of Slr2111/AMP is lower than that of Slr2111/ATP means that while AMP binds to Slr2111 the inhibition becomes stronger,when ATP binds to Slr2111the inhibition becomes weaker.While AMP binds to Slr2111,due to the changes in conformation of Slr2111,Slr2111 binds more Slr1393g3,when,however,ATP binds to Slr2111,the complex stability of Slr1393g3 and Slr2111 decreased.Hence,while AMP makes the complex of Slr1393g3 and Slr2111 more stable and Slr2111/AMP shows stronger inhibition on the chromophorylation of Slr1393g3,ATP makes the complex weaker and give the chance for Slr1393g3 to bind PCB.So Slr2111 works through binding nucleotides and thereby regulates its Slr1393-binding affinity and in consequence,regulating also the chromophorylation of Slr1393,work as sensing and regulating the cellular energy status protein.