Crystal Structure of VpsR Revealed Novel Dimeric Architecture and c-di-GMP Binding Site: Mechanistic Implications in Oligomerization, ATPase Activity and DNA Binding

J Mol Biol. 2022 Jan 30;434(2):167354. doi: 10.1016/j.jmb.2021.167354. Epub 2021 Nov 10.


VpsR, the master regulator of biofilm formation in Vibrio cholerae, is an atypical NtrC1 type bEBP lacking residues essential for σ54-RNAP binding and REC domain phosphorylation. Moreover, transcription from PvpsL, a promoter of biofilm biosynthesis, has been documented in presence of σ70-RNAP/VpsR/c-di-GMP complex. It was proposed that c-di-GMP and VpsR together form an active transcription complex with σ70-RNAP. However, the impact of c-di-GMP imparted on VpsR that leads to transcription activation with σ70-RNAP remained elusive, largely due to the lack of the structure of VpsR and knowledge about c-di-GMP:VpsR interactions. In this direction we have solved the crystal structure of VpsRRA, containing REC and AAA+ domains, in apo, AMPPNP/GMPPNP and c-di-GMP bound states. Structures of VpsRRA unveiled distinctive REC domain orientation that leads to a novel dimeric association and noncanonical ATP/GTP binding. Moreover, we have demonstrated that at physiological pH VpsR remains as monomer having no ATPase activity but c-di-GMP imparted cooperativity to convert it to dimer with potent activity. Crystal structure of c-di-GMP:VpsRRA complex reveals that c-di-GMP binds near the C-terminal end of AAA+ domain. Trp quenching studies on VpsRR, VpsRA, VpsRRA, VpsRAD with c-di-GMP additionally demonstrated that c-di-GMP could potentially bind VpsRD. We propose that c-di-GMP mediated tethering of VpsRD with VpsRA could likely favor generating the specific protein-DNA architecture for transcription activation.

Keywords: ATPase activity; X-ray crystallography; biofilm; fluorescence quenching; vibrio cholerae.

MeSH terms

  • Adenosine Triphosphatases / chemistry
  • Adenosine Triphosphatases / metabolism*
  • Bacterial Proteins / metabolism
  • Binding Sites*
  • Biofilms
  • Crystallography, X-Ray
  • Cyclic GMP / analogs & derivatives*
  • DNA, Bacterial / metabolism*
  • Gene Expression Regulation, Bacterial
  • Models, Molecular
  • Phosphorylation
  • Promoter Regions, Genetic
  • Protein Conformation
  • Signal Transduction
  • Vibrio cholerae / genetics


  • Bacterial Proteins
  • DNA, Bacterial
  • VpsR protein, Vibrio cholerae
  • bis(3',5')-cyclic diguanylic acid
  • Adenosine Triphosphatases
  • Cyclic GMP