The c-di-GMP Binding Protein YcgR Controls Flagellar Motor Direction and Speed to Affect Chemotaxis by a “Backstop Brake” Mechanism

183

310

Interactions of proteins with low-molecular-weight ligands, such as metabolites, cofactors, and allosteric regulators, are important determinants of metabolism, gene regulation, and cellular homeostasis. Pharmaceuticals often target these interactions to interfere with regulatory pathways. We have developed a rapid, precise, and high-throughput method for quantitatively measuring protein-ligand interactions without the need to purify the protein when performed in cells with low background activity. This method, differential radial capillary action of ligand assay (DRaCALA), is based on the ability of dry nitrocellulose to separate the free ligand from bound protein-ligand complexes. Nitrocellulose sequesters proteins and bound ligand at the site of application, whereas free ligand is mobilized by bulk movement of the solvent through capillary action. We show here that DRaCALA allows detection of specific interactions between three nucleotides and their cognate binding proteins. DRaCALA allows quantitative measurement of the dissociation constant and the dissociation rate. Furthermore, DRaCALA can detect the expression of a cyclic-di-GMP (cdiGMP)-binding protein in whole-cell lysates of Escherichia coli, demonstrating the power of the method to bypass the prerequisite for protein purification. We have used DRaCALA to investigate cdiGMP signaling in 54 bacterial species from 37 genera and 7 eukaryotic species. These studies revealed the presence of potential cdiGMP-binding proteins in 21 species of bacteria, including 4 unsequenced species. The ease of obtaining metabolite-protein interaction data using the DRaCALA assay will facilitate rapid identification of protein-metabolite and protein-pharmaceutical interactions in a systematic and comprehensive approach.whole-cell assay I nteractions of low-molecular-weight ligands with protein receptors are critical in biological signaling both between cells and within individual cells. Examples of intercellular signaling mediated by small molecules include quorum signaling in bacteria, hormone and neurotransmitter responses in endocrine systems of animals, and auxin and abscisic acid regulation in plants (1). Intracellular signaling also involves regulatory protein-binding molecules, such as calcium and cyclic nucleotides [e.g., cAMP, cGMP, cyclic-di-GMP (cdiGMP)] (2, 3). In fact, nucleotide receptors are often targets for therapeutic intervention (4). Thus, these protein-small ligand interactions have important implications in modern drug design and use. Considering that many protein-ligand interaction pairs represent potential targets of pharmaceutical intervention in disease or agriculture, there is an urgent need to collect qualitative and quantitative data for such protein-ligand interactions in a highthroughput manner. Current efforts in metabolomics are directed at cataloging the presence of various metabolites through mass spectrometric analysis of biological samples (5-8). However, this approach lacks the ability to confirm interactions with protein partners, and...

Section: Dracala Detection Of Cdigmp-binding Proteins In Diverse Pro-mentioning

confidence: 99%

Differential radial capillary action of ligand assay for high-throughput detection of protein-metabolite interactions

Roelofs

Wang²,

Sintim

et al. 2011

183

310

“…First, because cdiG is found naturally in E. coli, cdiG-specific phosphodiesterases exist for its breakdown (32). The presence of these phosphodiesterases, and the absence of cdiA-or cAG-specific phosphodiesterases, could have led to the lower observed levels of cdiG in the cell lysate.…”

mentioning

confidence: 99%

Hybrid promiscuous (Hypr) GGDEF enzymes produce cyclic AMP-GMP (3′, 3′-cGAMP)

Hallberg

Wang

Wright

et al. 2016

104

Over 30 years ago, GGDEF domain-containing enzymes were shown to be diguanylate cyclases that produce cyclic di-GMP (cdiG), a second messenger that modulates the key bacterial lifestyle transition from a motile to sessile biofilm-forming state. Since then, the ubiquity of genes encoding GGDEF proteins in bacterial genomes has established the dominance of cdiG signaling in bacteria. However, the observation that proteobacteria encode a large number of GGDEF proteins, nearing 1% of coding sequences in some cases, raises the question of why bacteria need so many GGDEF enzymes. In this study, we reveal that a subfamily of GGDEF enzymes synthesizes the asymmetric signaling molecule cyclic AMP-GMP (cAG or 3′, 3′-cGAMP). This discovery is unexpected because GGDEF enzymes function as symmetric homodimers, with each monomer binding to one substrate NTP. Detailed analysis of the enzyme from Geobacter sulfurreducens showed it is a dinucleotide cyclase capable of switching the major cyclic dinucleotide (CDN) produced based on ATP-to-GTP ratios. We then establish through bioinformatics and activity assays that hybrid CDN-producing and promiscuous substrate-binding (Hypr) GGDEF enzymes are found in other deltaproteobacteria. Finally, we validated the predictive power of our analysis by showing that cAG is present in surface-grown Myxococcus xanthus. This study reveals that GGDEF enzymes make alternative cyclic dinucleotides to cdiG and expands the role of this widely distributed enzyme family to include regulation of cAG signaling.bacterial signaling | surface sensing | second messengers | cyclic dinucleotides | fluorescent biosensor

“…To alter gene expression or protein activity in response to external or internal signals, c-di-GMP interacts with several proteins (1,2,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). In addition, two classes of RNAs have been reported that bind this second messenger, the c-di-GMP-I (class I) and c-di-GMP-II (class II) riboswitches (18,19).…”

mentioning

confidence: 99%

Structural basis of differential ligand recognition by two classes of bis-(3′-5′)-cyclic dimeric guanosine monophosphate-binding riboswitches

Smith

Shanahan

Moore

et al. 2011

107

195

The bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling pathway regulates biofilm formation, virulence, and other processes in many bacterial species and is critical for their survival. Two classes of c-di-GMP-binding riboswitches have been discovered that bind this second messenger with high affinity and regulate diverse downstream genes, underscoring the importance of RNA receptors in this pathway. We have solved the structure of a c-di-GMP-II riboswitch, which reveals that the ligand is bound as part of a triplex formed with a pseudoknot. The structure also shows that the guanine bases of c-di-GMP are recognized through noncanonical pairings and that the phosphodiester backbone is not contacted by the RNA. Recognition is quite different from that observed in the c-di-GMP-I riboswitch, demonstrating that at least two independent solutions for RNA second messenger binding have evolved. We exploited these differences to design a c-di-GMP analog that selectively binds the c-di-GMP-II aptamer over the c-di-GMP-I RNA. There are several bacterial species that contain both types of riboswitches, and this approach holds promise as an important tool for targeting one riboswitch, and thus one gene, over another in a selective fashion.