Detection of Cyclic Dinucleotide Binding Proteins

  • Vincent T. LeeEmail author


Cyclic dinucleotides are a family of secondary messenger molecules that regulate bacterial physiology, cell division, motility, and biofilm formation. In response to stimuli, activated dinucleotide cyclases synthesize cyclic dinucleotides. Once made, cyclic dinucleotides bind macromolecule receptors, including proteins and RNA, to allosterically regulate downstream functions. Many important classes of cyclic di-GMP protein receptors have been identified including the PilZ domain, various degenerate enzymatic domains (GGDEF, EAL, and HD-GYP), the MshE domain, the AAA+ domain containing DNA binding proteins, as well as many unique examples. The identification of these cyclic di-GMP binding proteins and their cyclic di-GMP binding sites allows the generation of binding-defective alleles for interrogating the importance of cyclic di-GMP signaling in these regulated pathways. Using these tools, the field has revealed that cyclic di-GMP directly regulates many cellular functions through allosteric binding. Despite the success in the field of identifying protein receptors in the past few decades, cyclic dinucleotide receptors often can only be experimentally identified due to their diversity. To address these challenges, a number of experimental techniques have been utilized to empirically demonstrate interactions between cyclic dinucleotide and protein receptors. Here we will review the techniques used for the discovery and validation of these interactions by (1) affinity pull-down, (2) screening of proteins encoded by the genome, and (3) biochemical and structural methods. The use of these techniques will enable future development of predictive computational approaches that allow rapid identification and validation of cyclic dinucleotide receptor proteins. The identity of cyclic dinucleotide receptors will allow for a detailed understanding of the molecular mechanisms of cyclic dinucleotide signaling on cellular physiology.


Cyclic di-GMP Cyclic di-AMP Cyclic GMP-AMP Protein receptors 


  1. 1.
    Corrigan RM, Gründling A (2013) Cyclic di-AMP: another second messenger enters the fray. Nat Rev Microbiol 11:513–524. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Hengge R (2009) Principles of c-di-GMP signalling in bacteria. Nat Rev Microbiol 7:263–273CrossRefGoogle Scholar
  3. 3.
    Huynh TN, Woodward JJ (2016) Too much of a good thing: regulated depletion of c-di-AMP in the bacterial cytoplasm. Curr Opin Microbiol 30:22–29. CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Jenal U, Reinders A, Lori C (2017) Cyclic di-GMP: second messenger extraordinaire. Nat Rev Microbiol 15:271–284. CrossRefGoogle Scholar
  5. 5.
    Nelson JW, Breaker RR (2017) The lost language of the RNA World. Sci Signal 10:eaam8812. CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Römling U, Galperin MY, Gomelsky M (2013) Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 77:1–52. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Krasteva PV, Sondermann H (2017) Versatile modes of cellular regulation via cyclic dinucleotides. Nat Chem Biol 13:350–359. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Schirmer T, Jenal U (2009) Structural and mechanistic determinants of c-di-GMP signalling. Nat Rev Microbiol 7:724–735. nrmicro2203 [pii]CrossRefGoogle Scholar
  9. 9.
    Ramesh A (2015) Second messenger – sensing riboswitches in bacteria. Semin Cell Dev Biol 47–48:3–8. CrossRefPubMedGoogle Scholar
  10. 10.
    Smith KD, Strobel SA (2011) Interactions of the c-di-GMP riboswitch with its second messenger ligand. Biochem Soc Trans 39:647–651. BST0390647 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ross P et al (1987) Regulation of cellulose synthesis in Acetobacter xylinum by cyclic diguanylic acid. Nature 325:279–281CrossRefGoogle Scholar
  12. 12.
    Tal R et al (1998) Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. J Bacteriol 180:4416–4425CrossRefGoogle Scholar
  13. 13.
    Galperin MY, Gaidenko TA, Mulkidjanian AY, Nakano M, Price CW (2001a) MHYT, a new integral membrane sensor domain. FEMS Microbiol Lett 205:17–23CrossRefGoogle Scholar
  14. 14.
    Galperin MY, Nikolskaya AN, Koonin EV (2001b) Novel domains of the prokaryotic two-component signal transduction systems. FEMS Microbiol Lett 203:11–21CrossRefGoogle Scholar
  15. 15.
    Perlman RL, Pastan I (1969) Pleiotropic deficiency of carbohydrate utilization in an adenyl cyclase deficient mutant of Escherichia coli. Biochem Biophys Res Commun 37:151–157CrossRefGoogle Scholar
  16. 16.
    Imamura R, Yamanaka K, Ogura T, Hiraga S, Fujita N, Ishihama A, Niki H (1996) Identification of the cpdA gene encoding cyclic 3′,5′-adenosine monophosphate phosphodiesterase in Escherichia coli. J Biol Chem 271:25423–25429. CrossRefPubMedGoogle Scholar
  17. 17.
    Emmer M, deCrombrugghe B, Pastan I, Perlman R (1970) Cyclic AMP receptor protein of E. coli: its role in the synthesis of inducible enzymes. Proc Natl Acad Sci USA 66:480–487CrossRefGoogle Scholar
  18. 18.
    Zubay G, Schwartz D, Beckwith J (1970) Mechanism of activation of catabolite-sensitive genes: a positive control system. Proc Natl Acad Sci USA 66:104–110CrossRefGoogle Scholar
  19. 19.
    Kuo JF, Greengard P (1969) Cyclic nucleotide-dependent protein kinases. IV. Widespread occurrence of adenosine 3′,5′-monophosphate-dependent protein kinase in various tissues and phyla of the animal kingdom. Proc Natl Acad Sci USA 64:1349–1355CrossRefGoogle Scholar
  20. 20.
    Langan TA (1968) Histone phosphorylation: stimulation by adenosine 3′,5′-monophosphate. Science 162:579–580CrossRefGoogle Scholar
  21. 21.
    Miyamoto E, Kuo JF, Greengard P (1969) Cyclic nucleotide-dependent protein kinases. 3. Purification and properties of adenosine 3′,5′-monophosphate-dependent protein kinase from bovine brain. J Biol Chem 244:6395–6402PubMedGoogle Scholar
  22. 22.
    Walsh DA, Perkins JP, Krebs EG (1968) An adenosine 3′,5′-monophosphate-dependant protein kinase from rabbit skeletal muscle. J Biol Chem 243:3763–3765PubMedGoogle Scholar
  23. 23.
    Nirenberg M, Leder P (1964) RNA codewords and protein synthesis. The effect of trinucleotides upon the binding of sRNA to ribosomes. Science 145:1399–1407CrossRefGoogle Scholar
  24. 24.
    Gilman AG (1970) A protein binding assay for adenosine 3′:5′-cyclic monophosphate. Proc Natl Acad Sci USA 67:305–312CrossRefGoogle Scholar
  25. 25.
    Amikam D, Galperin MY (2006) PilZ domain is part of the bacterial c-di-GMP binding protein. Bioinformatics 22:3–6CrossRefGoogle Scholar
  26. 26.
    Morgan JL, McNamara JT, Zimmer J (2014) Mechanism of activation of bacterial cellulose synthase by cyclic di-GMP. Nat Struct Mol Biol 21:489–496. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Merighi M, Lee VT, Hyodo M, Hayakawa Y, Lory S (2007) The second messenger bis-(3′-5′)-cyclic-GMP and its PilZ domain-containing receptor Alg44 are required for alginate biosynthesis in Pseudomonas aeruginosa. Mol Microbiol 65:876–895Google Scholar
  28. 28.
    Whitney JC et al (2015) Dimeric c-di-GMP is required for post-translational regulation of alginate production in Pseudomonas aeruginosa. J Biol Chem 290:12451–12462. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Hickman JW, Harwood CS (2008) Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 69:376–389CrossRefGoogle Scholar
  30. 30.
    Matsuyama BY, Krasteva PV, Baraquet C, Harwood CS, Sondermann H, Navarro MV (2016) Mechanistic insights into c-di-GMP-dependent control of the biofilm regulator FleQ from Pseudomonas aeruginosa. Proc Natl Acad Sci USA 113:E209–E218. CrossRefPubMedGoogle Scholar
  31. 31.
    Lee VT, Matewish JM, Kessler JL, Hyodo M, Hayakawa Y, Lory S (2007) A cyclic-di-GMP receptor required for bacterial exopolysaccharide production. Mol Microbiol 65:1474–1484CrossRefGoogle Scholar
  32. 32.
    Duerig A et al (2009) Second messenger-mediated spatiotemporal control of protein degradation regulates bacterial cell cycle progression. Genes Dev 23:93–104. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Ryjenkov DA, Simm R, Römling U, Gomelsky M (2006) The PilZ domain is a receptor for the second messenger c-di-GMP: the PilZ domain protein YcgR controls motility in enterobacteria. J Biol Chem 281:30310–30314CrossRefGoogle Scholar
  34. 34.
    Boehm A et al (2010) Second messenger-mediated adjustment of bacterial swimming velocity. Cell 141:107–116. S0092-8674(10)00019-X [pii]CrossRefGoogle Scholar
  35. 35.
    Paul K, Nieto V, Carlquist WC, Blair DF, Harshey RM (2010) The c-di-GMP binding protein YcgR controls flagellar motor direction and speed to affect chemotaxis by a “backstop brake” mechanism. Mol Cell 38:128–139. S1097-2765(10)00200-5 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Navarro MV, Newell PD, Krasteva PV, Chatterjee D, Madden DR, O’Toole GA, Sondermann H (2011) Structural basis for c-di-GMP-mediated inside-out signaling controlling periplasmic proteolysis. PLoS Biol 9:e1000588. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Newell PD, Monds RD, O’Toole GA (2009) LapD is a bis-(3′,5′)-cyclic dimeric GMP-binding protein that regulates surface attachment by Pseudomonas fluorescens Pf0-1. Proc Natl Acad Sci USA 106:3461–3466CrossRefGoogle Scholar
  38. 38.
    Tschowri N, Schumacher MA, Schlimpert S, Chinnam NB, Findlay KC, Brennan RG, Buttner MJ (2014) Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development. Cell 158:1136–1147. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Jones CJ et al (2015) C-di-GMP regulates motile to sessile transition by modulating MshA pili biogenesis and near-surface motility behavior in Vibrio cholerae. PLoS Pathog 11:e1005068. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Roelofs KG et al (2015) Systematic identification of cyclic-di-GMP binding proteins in Vibrio cholerae reveals a novel class of cyclic-di-GMP-binding ATPases associated with type II secretion systems. PLoS Pathog 11:e1005232. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Wang YC et al (2016) Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain. Nat Commun 7:12481. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Corrigan RM, Campeotto I, Jeganathan T, Roelofs KG, Lee VT, Gründling A (2013) Systematic identification of conserved bacterial c-di-AMP receptor proteins. Proc Natl Acad Sci USA 110:9084–9089. 1300595110 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Sureka K et al (2014) The cyclic dinucleotide c-di-AMP is an allosteric regulator of metabolic enzyme function. Cell 158:1389–1401. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    McFarland AP et al (2017) Sensing of bacterial cyclic dinucleotides by the oxidoreductase RECON promotes NF-kappaB activation and shapes a proinflammatory antibacterial state. Immunity 46:433–445. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Duvel J et al (2012) A chemical proteomics approach to identify c-di-GMP binding proteins in Pseudomonas aeruginosa. J Microbiol Methods 88:229–236. CrossRefPubMedGoogle Scholar
  46. 46.
    Laventie BJ, Glatter T, Jenal U (2017) Pull-down with a c-di-GMP-specific capture compound coupled to mass spectrometry as a powerful tool to identify novel effector proteins. Methods Mol Biol (Clifton, NJ) 1657:361–376. CrossRefGoogle Scholar
  47. 47.
    Laventie BJ, Nesper J, Ahrne E, Glatter T, Schmidt A, Jenal U (2015) Capture compound mass spectrometry – a powerful tool to identify novel c-di-GMP effector proteins. J Vis Exp.
  48. 48.
    Steiner S, Lori C, Boehm A, Jenal U (2013) Allosteric activation of exopolysaccharide synthesis through cyclic di-GMP-stimulated protein-protein interaction. EMBO J 32:354–368. CrossRefPubMedGoogle Scholar
  49. 49.
    Orr MW, Lee VT (2017) Differential radial capillary action of ligand assay (DRaCALA) for high-throughput detection of protein-metabolite interactions in bacteria. Methods Mol Biol (Clifton, NJ) 1535:25–41. CrossRefGoogle Scholar
  50. 50.
    Roelofs KG, Wang J, Sintim HO, Lee VT (2011) Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions. Proc Natl Acad Sci USA 108:15528–15533. 1018949108 [pii]CrossRefPubMedGoogle Scholar
  51. 51.
    Fang X et al (2014) GIL, a new c-di-GMP-binding protein domain involved in regulation of cellulose synthesis in enterobacteria. Mol Microbiol 93:439–452. CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Orr MW, Donaldson GP, Severin GB, Wang J, Sintim HO, Waters CM, Lee VT (2015) Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc Natl Acad Sci USA 112:E5048–E5057. CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Corrigan RM, Bellows LE, Wood A, Gründling A (2016) ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria. Proc Natl Acad Sci USA 113:E1710–E1719. CrossRefPubMedGoogle Scholar
  54. 54.
    Hendrick WA, Orr MW, Murray SR, Lee VT, Melville SB (2017) Cyclic di-GMP binding by an assembly ATPase (PilB2) and control of type IV pilin polymerization in the Gram-positive pathogen Clostridium perfringens. J Bacteriol 199:e00034-17. CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Laventie BJ et al (2019) A surface-induced asymmetric program promotes tissue colonization by Pseudomonas aeruginosa. Cell Host Microbe 25:140–152.e6. CrossRefGoogle Scholar
  56. 56.
    Christen M, Christen B, Folcher M, Schauerte A, Jenal U (2005) Identification and characterization of a cyclic di-GMP-specific phosphodiesterase and its allosteric control by GTP. J Biol Chem 280:30829–30837. M504429200 [pii]CrossRefGoogle Scholar
  57. 57.
    Hickman JW, Tifrea DF, Harwood CS (2005) A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci USA 102:14422–14427CrossRefGoogle Scholar
  58. 58.
    Corrigan RM, Abbott JC, Burhenne H, Kaever V, Gründling A (2011) c-di-AMP is a new second messenger in Staphylococcus aureus with a role in controlling cell size and envelope stress. PLoS Pathog 7:e1002217. CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Greenberg JR (1979) Ultraviolet light-induced crosslinking of mRNA to proteins. Nucleic Acids Res 6:715–732CrossRefGoogle Scholar
  60. 60.
    Christen B et al (2006) Allosteric control of cyclic di-GMP signaling. J Biol Chem 281:32015–32024. CrossRefGoogle Scholar
  61. 61.
    Christen M, Christen B, Allan MG, Folcher M, Jeno P, Grzesiek S, Jenal U (2007) DgrA is a member of a new family of cyclic diguanosine monophosphate receptors and controls flagellar motor function in Caulobacter crescentus. Proc Natl Acad Sci USA 104:4112–4117. 0607738104 [pii]CrossRefPubMedGoogle Scholar
  62. 62.
    Pratt JT, Tamayo R, Tischler AD, Camilli A (2007) PilZ domain proteins bind cyclic diguanylate and regulate diverse processes in Vibrio cholerae. J Biol Chem 282:12860–12870CrossRefGoogle Scholar
  63. 63.
    Donaldson GP, Roelofs KG, Luo Y, Sintim HO, Lee VT (2012) A rapid assay for affinity and kinetics of molecular interactions with nucleic acids. Nucleic Acids Res 40(7):e48. gkr1299 [pii]CrossRefPubMedGoogle Scholar
  64. 64.
    Baraquet C, Harwood CS (2013) Cyclic diguanosine monophosphate represses bacterial flagella synthesis by interacting with the Walker A motif of the enhancer-binding protein FleQ. Proc Natl Acad Sci USA 110:18478–18483. CrossRefPubMedGoogle Scholar
  65. 65.
    Benach J et al (2007) The structural basis of cyclic diguanylate signal transduction by PilZ domains. EMBO J 26:5153–5166CrossRefGoogle Scholar
  66. 66.
    Chin KH et al (2010) The cAMP receptor-like protein CLP is a novel c-di-GMP receptor linking cell-cell signaling to virulence gene expression in Xanthomonas campestris. J Mol Biol 396:646–662. S0022-2836(09)01474-0 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Whitney JC, Colvin KM, Marmont LS, Robinson H, Parsek MR, Howell PL (2012) Structure of the cytoplasmic region of PelD, a degenerate diguanylate cyclase receptor that regulates exopolysaccharide production in Pseudomonas aeruginosa. J Biol Chem 287:23582–23593. CrossRefPubMedPubMedCentralGoogle Scholar
  68. 68.
    Duvel J et al (2016) Application of synthetic peptide arrays to uncover cyclic di-GMP binding motifs. J Bacteriol 198:138–146. CrossRefPubMedGoogle Scholar
  69. 69.
    Wang J et al (2011) Conservative change to the phosphate moiety of cyclic diguanylic monophosphate remarkably affects its polymorphism and ability to bind DGC, PDE, and PilZ proteins. J Am Chem Soc 133(24):9320–9330. CrossRefPubMedGoogle Scholar
  70. 70.
    Ramelot TA, Yee A, Cort JR, Semesi A, Arrowsmith CH, Kennedy MA (2007) NMR structure and binding studies confirm that PA4608 from Pseudomonas aeruginosa is a PilZ domain and a c-di-GMP binding protein. Proteins 66:266–271CrossRefGoogle Scholar
  71. 71.
    De N, Pirruccello M, Krasteva PV, Bae N, Raghavan RV, Sondermann H (2008) Phosphorylation-independent regulation of the diguanylate cyclase WspR. PLoS Biol 6:e67CrossRefGoogle Scholar
  72. 72.
    Navarro MV, De N, Bae N, Wang Q, Sondermann H (2009) Structural analysis of the GGDEF-EAL domain-containing c-di-GMP receptor FimX. Structure 17:1104–1116. S0969-2126(09)00252-4 [pii]CrossRefPubMedPubMedCentralGoogle Scholar
  73. 73.
    Tao F, He YW, Wu DH, Swarup S, Zhang LH (2010) The cyclic nucleotide monophosphate domain of Xanthomonas campestris global regulator Clp defines a new class of cyclic di-GMP effectors. J Bacteriol 192:1020–1029. JB.01253-09 [pii]CrossRefGoogle Scholar
  74. 74.
    Krasteva PV, Fong JC, Shikuma NJ, Beyhan S, Navarro MV, Yildiz FH, Sondermann H (2010) Vibrio cholerae VpsT regulates matrix production and motility by directly sensing cyclic di-GMP. Science 327:866–868. CrossRefPubMedPubMedCentralGoogle Scholar
  75. 75.
    Witte G, Hartung S, Buttner K, Hopfner KP (2008) Structural biochemistry of a bacterial checkpoint protein reveals diadenylate cyclase activity regulated by DNA recombination intermediates. Mol Cell 30:167–178. S1097-2765(08)00166-4 [pii]CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  1. 1.Department of Cell Biology and Molecular GeneticsUniversity of MarylandCollege ParkUSA

Personalised recommendations