Nucleotide binding by the widespread high-affinity cyclic di-GMP receptor MshEN domain

C-di-GMP is a bacterial second messenger regulating various cellular functions. Many bacteria contain c-di-GMP-metabolizing enzymes but lack known c-di-GMP receptors. Recently, two MshE-type ATPases associated with bacterial type II secretion system and type IV pilus formation were shown to specifically bind c-di-GMP. Here we report crystal structure of the MshE N-terminal domain (MshEN 1-145 ) from Vibrio cholerae in complex with c-di-GMP at a 1.37 Å resolution. This structure reveals a unique c-di-GMP-binding mode, featuring a tandem array of two highly conserved binding motifs, each comprising a 24-residue sequence RLGxx( L/V/I)(L/V/I )xx G ( L/V/I)(L/V/I )xxxxLxxxLxxQ that binds half of the c-di-GMP molecule, primarily through hydrophobic interactions. Mutating these highly conserved residues markedly reduces c-di-GMP binding and biofilm formation by V. cholerae . This c-di-GMP-binding motif is present in diverse bacterial proteins exhibiting binding affinities ranging from 0.5 μM to as low as 14 nM. The MshEN domain contains the longest nucleotide-binding motif reported to date. Cyclic-di-GMP is a bacterial second messenger that binds to the regulatory domain of ATPases of some bacteria. Here, the authors report the crystal structure of this interaction, identify a cyclic-di-GMP binding mode, and show that this interaction might be important for bacterial biofilm formation.