Structural and mechanistic determinants of c-di-GMP signalling

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

“…Each GGDEF domain brings a substrate molecule to the catalytic site (19,20). In the inhibited state, the photosensory modules apparently prevent enzymatic domains from forming a properly aligned homodimer, whereas light-induced conformational changes restore enzymatically productive domain alignment.…”

Section: Significancementioning

confidence: 99%

“…1). Further, in all DGCs whose regulation has been studied at the structural level, enzyme activation has been shown or predicted to occur via alignment of the rigid GGDEF domains rather than via intradomain conformational changes (19,20). Based on these considerations, we reasoned that bacteriophytochromes can regulate diverse output activities that depend on proper alignment of the two output domains (24).…”

Section: Significancementioning

confidence: 99%

Engineering adenylate cyclases regulated by near-infrared window light

Ryu

Kang

Nelson

et al. 2014

108

114

Bacteriophytochromes sense light in the near-infrared window, the spectral region where absorption by mammalian tissues is minimal, and their chromophore, biliverdin IXα, is naturally present in animal cells. These properties make bacteriophytochromes particularly attractive for optogenetic applications. However, the lack of understanding of how light-induced conformational changes control output activities has hindered engineering of bacteriophytochromebased optogenetic tools. Many bacteriophytochromes function as homodimeric enzymes, in which light-induced conformational changes are transferred via α-helical linkers to the rigid output domains. We hypothesized that heterologous output domains requiring homodimerization can be fused to the photosensory modules of bacteriophytochromes to generate light-activated fusions. Here, we tested this hypothesis by engineering adenylate cyclases regulated by light in the near-infrared spectral window using the photosensory module of the Rhodobacter sphaeroides bacteriophytochrome BphG1 and the adenylate cyclase domain from Nostoc sp. CyaB1. We engineered several light-activated fusion proteins that differed from each other by approximately one or two α-helical turns, suggesting that positioning of the output domains in the same phase of the helix is important for light-dependent activity. Extensive mutagenesis of one of these fusions resulted in an adenylate cyclase with a sixfold photodynamic range. Additional mutagenesis produced an enzyme with a more stable photoactivated state. When expressed in cholinergic neurons in Caenorhabditis elegans, the engineered adenylate cyclase affected worm behavior in a light-dependent manner. The insights derived from this study can be applied to the engineering of other homodimeric bacteriophytochromes, which will further expand the optogenetic toolset.phytochrome | protein engineering | adenylyl cyclase | cAMP | locomotion

“…The bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) signaling system is one key pathway that allows bacteria to alter their behavior in response to changing environmental conditions (reviewed in refs. [1][2][3][4]. Cyclicdi-GMP is a second messenger that is synthesized by diguanylate cyclases (DGCs) and degraded by specific phosphodiesterases in response to a variety of stimuli, including light, oxidative conditions, cell density, and antibiotics.…”

mentioning

confidence: 99%

“…Cyclicdi-GMP is a second messenger that is synthesized by diguanylate cyclases (DGCs) and degraded by specific phosphodiesterases in response to a variety of stimuli, including light, oxidative conditions, cell density, and antibiotics. Cyclic-di-GMP controls critical transitions such as the switch from a planktonic state to the formation of a biofilm (1,2). It also controls virulence in many pathogenic bacterial species (5,6).…”

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.