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

Abstract

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 (MshEN1-145) 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)xxG(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.

Versatile modes of cellular regulation via cyclic dinucleotides

“…Residues key for c-di-GMP recognition have been identified for some of these targets and the recognition mechanisms appear to differ significantly from that of the ATPase FleQ 59 . For example, recent work demonstrated that MshE binds c-di-GMP at its N-terminal domain primarily through hydrophobic interactions with a 24-residue-long RLGxx( L/V/I)(L/V/I )xx G ( L/V/I)(L/V/I )xxxxLxxxLxxQ sequence 67 .…”

The ins and outs of cyclic di-GMP signaling in Vibrio cholerae

“…A recent study showed that polymerization of MshA subunits is promoted by c-di-GMP binding via the ATPase MshE [17,28]. MshE binds c-di-GMP in its N-terminal domain [18,29]. Furthermore, structure of the MshE N-terminal domain revealed that c-di-GMP binds to the unique binding motifs consisting of a tandem array of two highly conserved 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ [29].…”

“…MshE binds c-di-GMP in its N-terminal domain [18,29]. Furthermore, structure of the MshE N-terminal domain revealed that c-di-GMP binds to the unique binding motifs consisting of a tandem array of two highly conserved 24-residue sequence RLGxx(L/V/I)(L/V/I)xxG(L/V/I)(L/V/I)xxxxLxxxLxxQ [29]. Binding to c-di-GMP activates MshE to polymerize the MshA pili, which facilitates attachment.…”

“…Binding to c-di-GMP activates MshE to polymerize the MshA pili, which facilitates attachment. Loss of mshE or mshA abolishes initial surface attachment, indicating that c-di-GMP-mediated synthesis of MshA pili is a critical early step in biofilm formation [29]. …”

AraC-like transcriptional activator CuxR binds c-di-GMP by a PilZ-like mechanism to regulate extracellular polysaccharide production

“…Similarly, production of alginate in mucoid Pseudomonas aeruginosa requires c-di-GMP binding to the PilZ domain in Alg44, which is part of the alginate synthase complex (9). In addition to PilZ domains, c-di-GMP can be recognized by other receptors including GGDEF domains with an I-site, enzymatically inactive EAL domains, GIL domain, the MshEN domain, and c-di-GMPspecific riboswitches (10)(11)(12)(13)(14)(15)(16)(17)(18). Moreover, several c-di-GMP receptors function as transcriptional regulators, belong to diverse protein families, and regulate various cellular functions.…”