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

VpsR, the master regulator of biofilm formation in Vibrio cholerae, is an atypical NtrC1 type bEBP lacking residues essential for σ-RNAP binding and REC domain phosphorylation. Moreover, transcription from P, a promoter of biofilm biosynthesis, has been documented in presence of σ-RNAP/VpsR/c-di-GMP complex. It was proposed that c-di-GMP and VpsR together form an active transcription complex with σ-RNAP. However, the impact of c-di-GMP imparted on VpsR that leads to transcription activation with σ-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 VpsR, containing REC and AAA domains, in apo, AMPPNP/GMPPNP and c-di-GMP bound states. Structures of VpsR 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:VpsR complex reveals that c-di-GMP binds near the C-terminal end of AAA domain. Trp quenching studies on VpsR, VpsR, VpsR, VpsR with c-di-GMP additionally demonstrated that c-di-GMP could potentially bind VpsR. We propose that c-di-GMP mediated tethering of VpsR with VpsR could likely favor generating the specific protein-DNA architecture for transcription activation.