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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.Some 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.

SUBMITTER: Dahlstrom KM

PROVIDER: S-EPMC4959289 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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

Dahlstrom Kurt M KM Giglio Krista M KM Sondermann Holger H O'Toole George A GA

Journal of bacteriology 20160513 11

<h4>Unlabelled</h4>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 a ...[more]

PMID: 27002135

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Project description:The bacterial intracellular second messenger, cyclic dimeric GMP (c-di-GMP), regulates biofilm formation for many bacteria. The binding of c-di-GMP by the inner membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbC's calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present, GcbC activity is significantly enhanced (~8-fold), indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the I-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of Pseudomonas fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.IMPORTANCE In many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP; however, it is unclear how undesired cross talk is mitigated in the context of this soluble signal and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-di-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the I-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a "cloud" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling, and biofilm formation.

Project description:UnlabelledWspR is a hybrid response regulator-diguanylate cyclase that is phosphorylated by the Wsp signal transduction complex in response to growth of Pseudomonas aeruginosa on surfaces. Active WspR produces cyclic di-GMP (c-di-GMP), which in turn stimulates biofilm formation. In previous work, we found that when activated by phosphorylation, yellow fluorescent protein (YFP)-tagged WspR forms clusters that are visible in individual cells by fluorescence microscopy. Unphosphorylated WspR is diffuse in cells and not visible. Thus, cluster formation is an assay for WspR signal transduction. To understand how and why WspR forms subcellular clusters, we analyzed cluster formation and the enzymatic activities of six single amino acid variants of WspR. In general, increased cluster formation correlated with increased in vivo and in vitro diguanylate cyclase activities of the variants. In addition, WspR specific activity was strongly concentration dependent in vitro, and the effect of the protein concentration on diguanylate cyclase activity was magnified when WspR was treated with the phosphor analog beryllium fluoride. Cluster formation appears to be an intrinsic property of phosphorylated WspR (WspR-P). These results support a model in which the formation of WspR-P subcellular clusters in vivo in response to a surface stimulus is important for potentiating the diguanylate cyclase activity of WspR. Subcellular cluster formation appears to be an additional means by which the activity of a response regulator protein can be regulated.ImportanceBacterial sensor proteins often phosphorylate cognate response regulator proteins when stimulated by an environmental signal. Phosphorylated response regulators then mediate an appropriate adaptive cellular response. About 6% of response regulator proteins have an enzymatic domain that is involved in producing or degrading cyclic di-GMP (c-di-GMP), a molecule that stimulates bacterial biofilm formation. In this work, we examined the in vivo and in vitro behavior of the response regulator-diguanylate cyclase WspR. When phosphorylated in response to a signal associated with surface growth, WspR has a tendency to form oligomers that are visible in cells as subcellular clusters. Our results show that the formation of phosphorylated WspR (WspR-P) subcellular clusters is important for potentiating the diguanylate cyclase activity of WspR-P, making it more active in c-di-GMP production. We conclude that oligomer formation visualized as subcellular clusters is an additional mechanism by which the activities of response regulator-diguanylate cyclases can be regulated.

Dataset Information

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

Publications

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

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