Structure of PP4397 reveals the molecular basis for different c-di-GMP binding modes by Pilz domain proteins

J Mol Biol. 2010 Apr 23;398(1):97-110. doi: 10.1016/j.jmb.2010.03.007. Epub 2010 Mar 10.


Cyclic diguanylate (c-di-GMP) is a global regulator that modulates pathogen virulence and biofilm formation in bacteria. Although a bioinformatic study revealed that PilZ domain proteins are the long-sought c-di-GMP binding proteins, the mechanism by which c-di-GMP regulates them is uncertain. Pseudomonas putida PP4397 is one such protein that contains YcgR-N and PilZ domains and the apo-PP4397 structure was solved earlier by the Joint Center for Structural Genomics. We determined the crystal structure of holo-PP4397 and found that two intercalated c-di-GMPs fit into the junction of its YcgR-N and PilZ domains. Moreover, c-di-GMP binding induces PP4397 to undergo a dimer-to-monomer transition. Interestingly, another PilZ domain protein, VCA0042, binds to a single molecule of c-di-GMP, and both its apo and holo forms are dimeric. Mutational studies and the additional crystal structure of holo-VCA0042 (L135R) showed that the Arg122 residue of PP4397 is crucial for the recognition of two molecules of c-di-GMP. Thus, PilZ domain proteins exhibit different c-di-GMP binding stoichiometry and quaternary structure, and these differences are expected to play a role in generating diverse forms of c-di-GMP-mediated regulation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Crystallography, X-Ray
  • Cyclic GMP / analogs & derivatives
  • Cyclic GMP / chemistry
  • Cyclic GMP / genetics
  • Cyclic GMP / metabolism
  • Dimerization
  • Guanosine Monophosphate / genetics
  • Guanosine Monophosphate / metabolism*
  • Hydrogen Bonding
  • Kinetics
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Weight
  • Protein Binding / genetics
  • Protein Structure, Secondary
  • Protein Structure, Tertiary*
  • Pseudomonas putida / genetics
  • Pseudomonas putida / metabolism
  • Sequence Homology, Amino Acid


  • Bacterial Proteins
  • bis(3',5')-cyclic diguanylic acid
  • Guanosine Monophosphate
  • Cyclic GMP

Associated data

  • PDB/3KYF
  • PDB/3KYG