The Inhibitory Site of a Diguanylate Cyclase Is a Necessary Element for Interaction and Signaling with an Effector Protein

Abstract

Many bacteria contain large cyclic diguanylate (c-di-GMP) signaling networks made of diguanylate cyclases (DGCs) and phosphodiesterases that can direct cellular activities sensitive to c-di-GMP levels. While DGCs synthesize c-di-GMP, many DGCs also contain an autoinhibitory site (I-site) that binds c-di-GMP to halt excess production of this small molecule, thus controlling the amount of c-di-GMP available to bind to target proteins in the cell. Many DGCs studied to date have also been found to signal for a specific c-di-GMP-related process, and although recent studies have suggested that physical interaction between DGCs and target proteins may provide this signaling fidelity, the importance of the I-site has not yet been incorporated into this model. Our results from Pseudomonas fluorescens indicate that mutation of residues at the I-site of a DGC disrupts the interaction with its target receptor. By creating various substitutions to a DGC's I-site, we show that signaling between a DGC (GcbC) and its target protein (LapD) is a combined function of the I-site-dependent protein-protein interaction and the level of c-di-GMP production. The dual role of the I-site in modulating DGC activity as well as participating in protein-protein interactions suggests caution in interpreting the function of the I-site as only a means to negatively regulate a cyclase. These results implicate the I-site as an important positive and negative regulatory element of DGCs that may contribute to signaling specificity. IMPORTANCESome bacteria contain several dozen diguanylate cyclases (DGCs), nearly all of which signal to specific receptors using the same small molecule, c-di-GMP. Signaling specificity in these networks may be partially driven by physical interactions between DGCs and their receptors, in addition to the autoinhibitory site of DGCs preventing the overproduction of c-di-GMP. In this study, we show that disruption of the autoinhibitory site of a DGC in Pseudomonas fluorescens can result in the loss of interactions with its target receptor and reduced biofilm formation, despite increased production of c-di-GMP. Our findings implicate the autoinhibitory site as both an important feature for signaling specificity through the regulation of c-di-GMP production and a necessary element for the physical interaction between a diguanylate cyclase and its receptor.A major method of intracellular signaling in bacteria revolves around the second messenger cyclic diguanylate (c-di-GMP). c-di-GMP is produced by diguanylate cyclases (DGCs) and degraded by phosphodiesterases (PDEs) in networks that may range from one or a few such proteins to several dozen (1). Effector proteins then sense c-di-GMP by binding it and promote some specific cellular action as a result. Importantly, many DGCs studied to date promote only one or a small number of such processes (2-6), indicating that there must be mechanisms in place that allow for specificity in signaling among the many members of a c-di-GMP network.A key regulatory element in signalin...

Versatile modes of cellular regulation via cyclic dinucleotides

“…The first structure of a DGC, PleD from C. crescentus , also revealed the so-called inhibitory (I−) site as prevalent among GGDEF domain-containing proteins 22 (Figure 1b). A conserved I-site RxxD motif partakes in a secondary c-di-GMP binding site, that is distal from the enzyme’s active site and can be involved in negative-feedback regulation and/or protein-protein interactions 22,23 . For c-di-GMP degradation, β-barrel EAL or the unrelated HD-GYP domains confer specific PDE activity to proteins 24,25 .…”

“…To date, several examples of inactive EAL domains binding c-di-GMP at their degenerate active sites have been described as signal transduction modules 35,36 . The I-site on GGDEF domains, on the other hand, can not only serve for feedback inhibition in the case of active enzymes, but can also provide a mechanism for c-di-GMP sensing and/or signal transmission in both degenerate and active DGCs 23,37,38 . Bioinformatics studies have also pinpointed PilZ domains as bona fide c-di-GMP sensors based on a phyletic distribution similar to those of GGDEF and EAL modules and a likely role in c-di-GMP mediated processes 39 .…”

Progress in Understanding the Molecular Basis Underlying Functional Diversification of Cyclic Dinucleotide Turnover Proteins

“…One of the hallmarks of cyclic di-GMP signaling is a relative or absolute specificity of a phenotypic output of an individual chromosomally encoded GGDEF domain protein. This specificity is partly explained by the close proximity of signal production/degradation with receptor and/or effector proteins mediated through protein-protein interactions, a first example being the involvement of the I-site of a GGDEF domain in interaction with an EAL domain cyclic di-GMP receptor (21,33). Interactions between the EAL domain protein YciR and diguanylate cyclase YdaM control a key step in E. coli biofilm formation through a suggested modulation of localized cyclic di-GMP levels (34).…”

“…The I-site, which is formed at an intra-or intermolecular interface bridged by a cyclic di-GMP dimer, variably extends beyond the central conserved RXXD cyclic di-GMP binding motif and mediates allosteric noncompetitive product inhibition, through feedback control of cyclic di-GMP synthesis (20,21). The RXXD motif is absent in a proportion of GGDEF domains; alternative mechanisms to control cyclic di-GMP synthesis have been described for some of these proteins (6,22,23).…”

“…The RXXD motif is absent in a proportion of GGDEF domains; alternative mechanisms to control cyclic di-GMP synthesis have been described for some of these proteins (6,22,23). A second recently discovered function of the I-site is the participation in protein-protein interaction with a cyclic di-GMP receptor, which ensures a stringent specificity of cyclic di-GMP signaling even in the presence of cyclic di-GMP production (21). In divergent GGDEF domain proteins (see below), a retained I-site in catalytically nonfunctional GGDEF domains converts these domains into cyclic di-GMP receptors (24)(25)(26)(27).…”