Structural basis of ligand binding by a c-di-GMP riboswitch

Department of Chemistry, Yale University, New Haven, Connecticut, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 11/2009; 16(12):1218-23. DOI: 10.1038/nsmb.1702
Source: PubMed


The second messenger signaling molecule bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) regulates many processes in bacteria, including motility, pathogenesis and biofilm formation. c-di-GMP-binding riboswitches are important downstream targets in this signaling pathway. Here we report the crystal structure, at 2.7 A resolution, of a c-di-GMP riboswitch aptamer from Vibrio cholerae bound to c-di-GMP, showing that the ligand binds within a three-helix junction that involves base-pairing and extensive base-stacking. The symmetric c-di-GMP is recognized asymmetrically with respect to both the bases and the backbone. A mutant aptamer was engineered that preferentially binds the candidate signaling molecule c-di-AMP over c-di-GMP. Kinetic and structural data suggest that genetic regulation by the c-di-GMP riboswitch is kinetically controlled and that gene expression is modulated through the stabilization of a previously unidentified P1 helix, illustrating a direct mechanism for c-di-GMP signaling.

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    • "The ability of a second messenger to have numerous effects on cellular behavior lies in the diversity of c-di-GMP receptors. In other species of bacteria, c-di-GMP has been demonstrated to exert its regulatory effects through proteins with cyclic nucleotide monophosphate domains (Tao et al., 2010), ribonucleoprotein complexes (Tuckerman et al., 2011), transcriptional regulators (Hickman and Harwood, 2008; Krasteva et al., 2010; Fazli et al., 2011), GEMM riboswitches (Sudarsan et al., 2008; Smith et al., 2009; Luo et al., 2013), and PilZ domain-containing proteins (Amikam and Galperin, 2006; Ryjenkov et al., 2006; Bian et al., 2013). To date, only one c-di-GMP-binding protein, the PilZ domain-containing protein PlzA, has been identified in B. burgdorferi (Freedman et al., 2010; Pitzer et al., 2011). "
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    ABSTRACT: In nature, the Lyme disease spirochete Borrelia burgdorferi cycles between the unrelated environments of the Ixodes tick vector and mammalian host. In order to survive transmission between hosts, B. burgdorferi must be able to not only detect changes in its environment, but also rapidly and appropriately respond to these changes. One manner in which this obligate parasite regulates and adapts to its changing environment is through cyclic-di-GMP (c-di-GMP) signaling. c-di-GMP has been shown to be instrumental in orchestrating the adaptation of B. burgdorferi to the tick environment. B. burgdorferi possesses only one set of c-di-GMP-metabolizing genes (one diguanylate cyclase and two distinct phosphodiesterases) and one c-di-GMP-binding PilZ-domain protein designated as PlzA. While studies in the realm of c-di-GMP signaling in B. burgdorferi have exploded in the last few years, there are still many more questions than answers. Elucidation of the importance of c-di-GMP signaling to B. burgdorferi may lead to the identification of mechanisms that are critical for the survival of B. burgdorferi in the tick phase of the enzootic cycle as well as potentially delineate a role (if any) c-di-GMP may play in the transmission and virulence of B. burgdorferi during the enzootic cycle, thereby enabling the development of effective drugs for the prevention and/or treatment of Lyme disease.
    Full-text · Article · May 2014 · Frontiers in Cellular and Infection Microbiology
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    • "The cyclic-di-GMP aptamer, for example, binds its ligand with a Kd of ∼10 pM. It achieves such tight binding because its dissociation rate is extremely slow (t1/2 ∼ 43 days) (35). As E. coli grown in rich medium divide approximately every 20 min and the average mRNA half-life is ∼6 min (36), this riboswitch cannot reach equilibrium, and thus acts as a kinetic trap like the theophylline riboswitches described here. "
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    ABSTRACT: Riboswitches are sequences of RNA that control gene expression via RNA–ligand interactions, without the need for accessory proteins. Riboswitches consist of an aptamer that recognizes the ligand and an expression platform that couples ligand binding to a change in gene expression. Using in vitro selection, it is possible to screen large (∼1013 members) libraries of RNA sequences to discover new aptamers. However, limitations in bacterial transformation efficiency make screening such large libraries for riboswitch function in intact cells impractical. Here we show that synthetic riboswitches function in an E. coli S30 extract in a manner similar to how they function in intact E. coli cells. We discovered that, although this family of riboswitches regulates the initiation of protein translation, the fate of whether an RNA message is translated is determined during transcription. Thus, ligand binding does not bias a population of rapidly equilibrating RNA structures, but rather, co-transcriptional ligand binding kinetically traps the RNA in a conformation that supports efficient translation. In addition to providing new insights into the mechanisms of action of a family of synthetic riboswitches, our experiments suggest that it may be possible to perform selections for novel synthetic riboswitches in an in vitro system.
    Full-text · Article · Apr 2014 · Nucleic Acids Research
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    • "In the cis form, the two bases of the monomer are rotated 90° resulting in partially overlapping of the guanine bases (Fig. 1B). The cis-monomer binds both VCA0042 [13], a PilZ-domain containing protein, as well as RNA riboswitches, such as Vc2 and GEMM [14], [15], [16]. In the cis rotaform, cdiGMP can self-assemble to form higher order complexes [17], [18], [19]. "
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    ABSTRACT: Bacterial signaling networks control a wide variety of cellular processes including growth, metabolism, and pathogenesis. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (cdiGMP) is a secondary signaling nucleotide that controls cellulose synthesis, biofilm formation, motility and virulence in a wide range of Gram-negative bacterial species. CdiGMP is a dynamic molecule that forms different tertiary structures in vitro, including a trans-monomer, cis-monomer, cis-dimer and G-octaplex (G8). Although the monomer and dimer have been shown to be physiologically relevant in modulating protein activity and transcription, the biological effects of the cdiGMP G8 has not yet been described. Here, we have developed a TLC-based assay to detect radiolabeled cdiGMP G8 formation. Utilizing the radiolabeled cdiGMP G8, we have also shown a novel inhibitory interaction between the cdiGMP G8 and HIV-1 reverse transcriptase and that the cdiGMP G8 does not interact with proteins from Pseudomonas aeruginosa known to bind monomeric and dimeric cdiGMP. These results suggest that the radiolabeled cdiGMP G8 can be used to measure interactions between the cdiGMP G8 and cellular proteins, providing an avenue through which the biological significance of this molecule could be investigated.
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