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c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry–related pockets

Nature Chemical Biology volume 10, pages 780–786 (2014)Cite this article

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Abstract

The ydaO riboswitch, involved in sporulation, osmotic stress responses and cell wall metabolism, targets the second messenger cyclic-di-AMP with subnanomolar affinity. We have solved the structure of c-di-AMP bound to the Thermoanaerobacter tengcongensis ydaO riboswitch, thereby identifying a five-helical scaffold containing a zippered-up bubble, a pseudoknot and long-range tertiary base pairs. Highlights include the identification of two c-di-AMP binding pockets on the same face of the riboswitch, related by pseudo-two-fold symmetry, with potential for cross-talk between sites mediated by adjacently positioned base-stacking alignments connecting pockets. The adenine rings of bound c-di-AMP molecules are wedged between bases and stabilized by stacking, base-sugar and sugar-sugar intermolecular hydrogen bonding interactions. The structural studies are complemented by isothermal titration calorimetry–based binding studies of mutants mediating key tertiary intermolecular contacts. The T. tengcongensis ydaO riboswitch, like its Bacillus subtilis counterpart, most likely functions through a transcription termination mechanism, with the c-di-AMP bound state representing an 'off' switch.

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Figure 1: Secondary and tertiary structure of the T. tengcongensis ydaO riboswitch bound to c-di-AMP and alignment of c-di-AMP molecules A and B in their respective binding pockets.
Figure 2: Folding topologies adopted by the bubble and pseudoknot segments and their flanking stem segments in the structure of T. tengcongensis ydaO riboswitch bound to c-di-AMP.
Figure 3: Similarities in recognition features between Aα rings of c-di-AMP molecules A and B bound within their respective binding pockets and a pair of parallel base stacking alignments connecting binding pockets in the structure of T. tengcongensis ydaO riboswitch bound to c-di-AMP.
Figure 4: Comparison of ITC binding curves with other second messengers and analogs and mutants involved in tertiary pairing in the T. tengcongensis ydaO riboswitch.
Figure 5: Comparison of binding pockets and intermolecular hydrogen bonding contacts in complexes of c-di-AMP bound to the ydaO riboswitch and the protein DisA.

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Acknowledgements

We acknowledge assistance by staff at the X-29 beamline at the Brookhaven National Laboratory and NE-CAT beamlines at the Advanced Photon Source. We thank B. Gaffney and R. Jones of Rutgers University for samples of chemically synthesized cGAMP linkage isomers. The research was supported by US National Institutes of Health grant 1 U19 CA179564 to D.J.P.

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  1. Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

    Aiming Ren & Dinshaw J Patel

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A.R. undertook all of the crystallographic and ITC experiments under the supervision of D.J.P.

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Correspondence to Dinshaw J Patel.

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Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–19. (PDF 38749 kb)

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Ren, A., Patel, D. c-di-AMP binds the ydaO riboswitch in two pseudo-symmetry–related pockets. Nat Chem Biol 10, 780–786 (2014). https://doi.org/10.1038/nchembio.1606

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