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

Nature Structural & Molecular Biology volume 16, pages 1218–1223 (2009)Cite this article

Abstract

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 Å 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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Figure 1: Structure of the c-di-GMP riboswitch aptamer.
Figure 2: Recognition of c-di-GMP by the riboswitch.
Figure 3: Affinity and rate measurements for c-di-GMP binding to wild-type and mutant riboswitches.

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Acknowledgements

We thank S. Myers and the beamline staff at X25, X12C and X29 at the National Synchrotron Light Source at Brookhaven National Laboratory; M. Strickler, D. Keller, and the Yale Center for Structural Biology core staff; Y. Xiong for data processing help and advice; C. Shanahan, E. Butler, N. Carrasco, J. Cochrane and M. Stahley for help and advice; D. Hiller, J. Davis, E. Lee, N. Sudarsan and other members of the Strobel and Breaker labs for helpful discussions; V. Singh (Strobel lab) for synthesis of iridium hexammine; U. Jenal (University of Basel) for the gift of the PleD* expression plasmid; K.-P. Hopfner (University of Munich) for the gift of the DisA expression plasmid; and G. Reguera (Michigan State University) and B. Bassler (Princeton University) for the gift of V. cholerae genomic DNA. This work was supported by the US National Institutes of Health grant GM02278 and the National Science Foundation grant MCB0544255 to S.A.S.

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Authors and Affiliations

  1. Department of Chemistry, Yale University, New Haven, Connecticut, USA

    Kathryn D Smith, Sarah V Lipchock & Scott A Strobel

  2. Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut, USA

    Tyler D Ames & Ronald R Breaker

  3. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

    Jimin Wang, Ronald R Breaker & Scott A Strobel

  4. Howard Hughes Medical Institute, Yale University, New Haven, Connecticut, USA

    Ronald R Breaker

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  1. Kathryn D Smith
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Contributions

K.D.S. determined the crystal structure, performed biochemical experiments and wrote the manuscript. S.V.L. performed biochemical experiments. T.D.A. performed the updated sequence alignment and performed the in-line probing experiments. J.W. gave critical help in data processing and refinement. R.R.B. and S.A.S. advised on the project. All authors discussed the data and provided comments on the manuscript.

Corresponding author

Correspondence to Scott A Strobel.

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Competing interests

R.R.B. is a cofounder of BioRelix, a biotechnology company pursuing the development of riboswitches as antibiotics targets.

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Supplementary Figures 1–7, Supplementary Data/Discussion and Supplementary Methods (PDF 4959 kb)

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Smith, K., Lipchock, S., Ames, T. et al. Structural basis of ligand binding by a c-di-GMP riboswitch. Nat Struct Mol Biol 16, 1218–1223 (2009). https://doi.org/10.1038/nsmb.1702

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