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Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography

Nature volume 541, pages 242–246 (2017)Cite this article

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Abstract

Riboswitches are structural RNA elements that are generally located in the 5′ untranslated region of messenger RNA. During regulation of gene expression, ligand binding to the aptamer domain of a riboswitch triggers a signal to the downstream expression platform1,2,3. A complete understanding of the structural basis of this mechanism requires the ability to study structural changes over time4. Here we use femtosecond X-ray free electron laser (XFEL) pulses5,6 to obtain structural measurements from crystals so small that diffusion of a ligand can be timed to initiate a reaction before diffraction. We demonstrate this approach by determining four structures of the adenine riboswitch aptamer domain during the course of a reaction, involving two unbound apo structures, one ligand-bound intermediate, and the final ligand-bound conformation. These structures support a reaction mechanism model with at least four states and illustrate the structural basis of signal transmission. The three-way junction and the P1 switch helix of the two apo conformers are notably different from those in the ligand-bound conformation. Our time-resolved crystallographic measurements with a 10-second delay captured the structure of an intermediate with changes in the binding pocket that accommodate the ligand. With at least a 10-minute delay, the RNA molecules were fully converted to the ligand-bound state, in which the substantial conformational changes resulted in conversion of the space group. Such notable changes in crystallo highlight the important opportunities that micro- and nanocrystals may offer in these and similar time-resolved diffraction studies. Together, these results demonstrate the potential of ‘mix-and-inject’ time-resolved serial crystallography to study biochemically important interactions between biomacromolecules and ligands, including those that involve large conformational changes.

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Figure 1: Structure comparison of the ligand-bound and apo conformers.
Figure 2: Structures of the three-way junction in the absence of ligand.
Figure 3: Setup of mix-and-inject SFX and conversion of the structure and crystal lattice.
Figure 4: Visualizing the ligand-bound intermediate state.

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Acknowledgements

Portions of this research were carried out at the Linac Coherent Light Source, a National User Facility operated by Stanford University on behalf of the US Department of Energy, Office of Basic Energy Sciences. The CXI instrument was funded by the LCLS Ultrafast Science Instruments (LUSI) project funded by the US Department of Energy, Office of Basic Energy Sciences. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. We thank J. Strathern and M. Dunne for their support and S. Wakatsuki for discussions. This work is supported in part by the NSF-STC “BioXFEL” (NSF-1231306), the NIH Intramural Research Programs of NCI, CIT, NHLBI, and the US Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357, the European Research Council, “Frontiers in Attosecond X-ray Science: Imaging and Spectroscopy (AXSIS)”, ERC-2013-SyG 609920, and the BMBF through project 05K16GU1.

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Authors and Affiliations

  1. Protein-Nucleic Acid Interaction Section, Structural Biophysics Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, 21702, Maryland, USA

    J. R. Stagno, Y. Liu, Y. R. Bhandari, M. Swain, D. R. Wendel, R. A. Tuckey, P. Yu, M. Dyba, S. G. Tarasov & Y.-X. Wang

  2. Department of Biochemistry, Arizona State University, Tempe, 85287, Arizona, USA

    C. E. Conrad, J. D. Coe & P. Fromme

  3. Center for Applied Structural Discovery, The Biodesign Institute, Arizona State University, Tempe, 85287, Arizona, USA

    C. E. Conrad, J. D. Coe, U. Weierstall, P. Fromme, N. A. Zatsepin & J. C. H. Spence

  4. Department of Biophysics, Johns Hopkins University, Baltimore, 21218, Maryland, USA

    S. Panja & S. A. Woodson

  5. Small Angle X-ray Scattering Core Facility, Center for Cancer Research, National Cancer Institute, Frederick, 21702, Maryland, USA

    L. Fan

  6. Department of Physics, Arizona State University, Tempe, 85287, Arizona, USA

    G. Nelson, C. Li, U. Weierstall, N. A. Zatsepin & J. C. H. Spence

  7. Center for Free-Electron Laser Science, Deutsches Elektronen-Synchrotron DESY, Notkestraße 85, Hamburg, 22607, Germany

    T. A. White, M. O. Wiedorn, J. Knoska, D. Oberthuer, A. Barty & H. N. Chapman

  8. Department of Physics, University of Hamburg, Luruper Chaussee 149, Hamburg, 22607, Germany

    M. O. Wiedorn, J. Knoska & H. N. Chapman

  9. Hauptmann-Woodward Medical Research Institute, Buffalo, 14203, New York, USA

    T. D. Grant

  10. Center for Information Technology, National Institutes of Health, Bethesda, 20892-5624, Maryland, USA

    C. D. Schwieters

  11. Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, 20892, Maryland, USA

    J. Zhang

  12. Laboratory of RNA Biophysics and Cellular Physiology, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, 20892, Maryland, USA

    A. R. Ferré-D’Amaré

  13. Department of Chemistry, Johns Hopkins University, Baltimore, 21218, Maryland, USA

    D. E. Draper

  14. Linac Coherent Light Source, SLAC National Accelerator Laboratory, Menlo Park, 94025, California, USA

    M. Liang, M. S. Hunter & S. Boutet

  15. Biosciences Division, Structural Biology Center, Advanced Photon Source, Argonne National Laboratory, Argonne, 60439, Illinois, USA

    K. Tan

  16. X-ray Science Division, Advanced Photon Source, Argonne National Laboratory, Argonne, 60439, Illinois, USA

    X. Zuo

  17. Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute, Frederick, 21702, Maryland, USA

    X. Ji

Authors
  1. J. R. Stagno
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  2. Y. Liu
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  8. G. Nelson
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  9. C. Li
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  10. D. R. Wendel
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  23. J. Zhang
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  32. X. Ji
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  36. J. C. H. Spence
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  37. S. A. Woodson
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  38. Y.-X. Wang
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Contributions

J.R.S. and Y.-X.W. designed experiments; Y.L. and P.Y. provided the RNA samples; J.R.S. and Y.L. crystallized the RNA; J.R.S., Y.L., Y.R.B., D.R.W., C.E.C., J.D.C., G.N., C.L., N.A.Z., M.O.W., D.O., J.K., T.D.G., M.S.H., S.B., M.L. and Y.-X.W. collected the SFX data; M.O.W., D.O., J.K. and H.N.C. designed the mixing setup; J.R.S., Y.R.B., D.R.W., T.A.W., A.B., R.A.T., N.A.Z., X.J. and T.D.G. processed and analysed the SFX data; J.R.S., Y.R.B., M.S., X.J. and Y.-X.W. interpreted the SFX data; N.A.Z., A.B., M.S.H., S.B., M.L., U.W., P.F., H.N.C. and J.C.H.S. contributed the XFEL expertise and support; Y.R.B., L.F., X.Z., C.D.S. and Y.-X.W. collected, analysed and interpreted SAXS data; J.R.S., C.E.C., K.T. and Y.-X.W. characterized samples; M.D. and Y.L. performed mass spectroscopy; S.G.T. and Y.L. performed ITC and fluorescence titration; S.P. and Y.L. collected and analysed binding data; X.Z. and S.A.W. modelled ligand binding kinetics; X.Z., S.P., S.A.W. and Y.-X.W. interpreted kinetic data; D.E.D., A.F.D. and J.Z. contributed discussions; J.R.S. and Y.-X.W. drafted the manuscript and all authors contributed to the revision.

Corresponding author

Correspondence to Y.-X. Wang.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks J. Hajdu, R. Micura and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Characterization of RNA crystals.

Microcrystals of apo-rA71 were grown using batch crystallization as described in Methods. A SONICC Imager (Formulatrix) was used to image each sample (0.5–1.0 μl) of crystals using three different methods: visible light (a); UV-TPEF (b); and second-order nonlinear imaging of chiral crystals (SONICC) (c). d, Crystal samples were observed using a stereomicroscope (Zeiss) under cross-polarized light. e, Without cross-polarization, crystals were barely observable. f, The relative quality of the crystalline samples was measured by powder X-ray diffraction (APS beamline 19-ID), with a maximum observable resolution of approximately 6 Å. The resolution ring (red) corresponds to 6.8 Å.

Extended Data Figure 2 Characterization of rA71 in solution by small angle X-ray scattering.

a, Comparison of the Kratky plots of the solution X-ray scattering curves of rA71 in the apo (black) and bound (red) states. b, Plot of back-calculated small angle X-ray scattering (SAXS) profiles of apo1 and apo2 conformers along with solution X-ray scattering curves of rA71 in the apo (red) states. c, Experimental SAXS curve shown in red with red error bars and superimposed with the SAXS curves that were back-calculated from 128 structures using the two-member ensemble calculation. The ratio of the two conformers (apo2:apo1) is approximately 0.9:0.1 to give the best fit to the experiment data with χ2 values ranging approximately from 0.1 to 0.3.

Extended Data Figure 3 Three-way junction undergoes notable structural rearrangements to accommodate ligand, compressing the major groove.

a, The three-way junction, depicted in three orientations, as observed in the apo1 (blue), apo2 (cyan,), and ligand-bound (magenta, PDB code 4TZX) structures. Virtually all residues in the three-way junction undergo considerable conformational changes upon ligand binding. Most notable are the ‘swinging’ residues in the hinge (U22, A23) and latch (U48, U49, U51) regions, the atomic positions of which differ by as much as 17 Å in the apo conformers relative to the ligand-bound conformer. b, In the absence of ligand, concerted movement of the hinge (depicted as white surface and stick model) and latch regions results in considerable narrowing of the major groove formed between helices P1 and P3, which measures 9.4 Å, 10.0 Å and 16.6 Å for apo1, apo2 and ligand-bound conformers, respectively. Major groove distances were measured between the phosphorous atoms of U71 and A19 (or U20 in the case of apo1 owing to a difference in register).

Extended Data Figure 4 Multistage ligand binding kinetics to the rA71 riboswitch.

Ligand binding to the rA71 riboswitch was monitored by replacing U48 with the fluorescent base analogue 2-aminopurine (2AP; termed rA71-U482AP). The 2AP fluorescence emission intensity increases upon ligand-induced reorganization of the binding pocket. The 2AP substitution does not change the secondary structure of the apo rA71 riboswitch, as judged by partial RNase digestion. a, Equilibrium titration of rA71-U482AP with adenine yields Kd = 5 μM (black symbols). A similar Kd value was obtained from the endpoints of the kinetics progress curves (red symbols) and from in-line probing experiments. This value is about tenfold higher than adenine binding to the unmodified riboswitch, possibly because 2AP forms more stable base-stacking interactions in the apo structure. Error bars show the standard deviation from the average of two or more independent trials. b, The ligand-binding kinetics is consistent with a four-state mechanism. Binding of 0.5–1,600 μM adenine to 0.5 μM rA71-U482AP was measured by stopped-flow fluorescence (1.8 ms deadtime), as described in the Methods. The apparent rate constants for adenine association, λfast and λslow, were obtained from fits of individual trajectories to a biphasic rate equation, ∆F = Afast(1 − exp(−λfastt)) + Aslow(1 − exp(−λslowt)), where A denotes for adenine concentrations. Error bars as in a. The nonlinear increase in λfast (filled symbols) with adenine concentration over the full range of ligand concentrations indicates the presence of one or intermediates in the binding mechanism. The ligand-independent phase, λslow (open symbols), results in biphasic trajectories above 50 μM adenine and is explained by slow exchange between binding competent and binding incompetent forms of the riboswitch. c, The apparent bimolecular rate constant for adenine association is slower than diffusion and was obtained from λfast versus [adenine], under pseudo-first order conditions (0.5–25 μM adenine) in which ligand binding to the competent (open) riboswitch is rate-limiting. In 10 mM MgCl2 (filled symbols), kon = 1.9 × 105 M−1 s−1 and koff = 1.7 s−1. In 1.25 mM MgCl2 (open symbols), kon = 5.2 × 104 M−1 s−1 and koff = 2.1 s−1. The error bars are as in a with n = 3 over three independent trials. d, e, The same set of reduced experimental data in Fig. 2c was globally fit to simpler three-state kinetic mechanisms as described in the Supplementary Discussion. These models were not able to describe the solution binding kinetics over the full-range of ligand concentrations tested. Therefore, the four-state model in equation (1) is the simplest mechanism capable of describing the data. We do not exclude the possibility that the riboswitch samples additional apo states and intermediate complexes that may contribute to the robustness of the switch mechanism. d, Three-state mechanism with only one apo state. The parameters obtained from the fitting are: kon = 0.28 μM−1 s−1, koff = 37 s−1, kf = 103 s−1, kr = 5.1 s−1, sc = 2.03. Err(k, sc) = 0.053, where sc is scaling value and rate constants (k) by minimizing a Chi-squared error function Err(k, sc) that describes the discrepancy between calculated curves and experimental data sets (Supplementary Discussion). e, Three-state mechanism with two apo states and no binding intermediate. Parameters: kop = 2.5 s−1, kcl = 0.58 s−1, kon = 0.16 μM−1 s−1, koff = 0.79 s−1, sc = 2.44. Err(k,sc) = 0.056.

Extended Data Figure 5 Time-course simulation of the IB concentration in the crystal and comparison of the unit cell dimensions of the apo, IB and bound structures.

a, Simulated time courses of the IB buildup and changes in concentrations of ligand, apo2 and bound (B) states in the crystal. See also Methods. b, c, Space group and unit-cell dimensions of the crystals of apo, IB and bound states. The structure was converted in the crystal from the apo to the bound state after at least 10 min of mixing with adenine ligand. The crystal lattice remains unchanged after 10 s of mixing with ligand.

Extended Data Figure 6 Determining the structure of the IB conformer.

a, To first verify whether there were changes in the IB state relative to apo2, the apo2 structure was refined against the 10-s-mix data; 2 Fo − Fc (1σ, blue) and Fo − Fc (3σ, green) electron density maps are shown. Both maps indicated alternative positions for both U48 and A21. b, The occupancies of U48 and A21 of the apo2 state were set to 0.5 and refined in the same manner. The Fo − Fc map (3σ, green) clearly indicated the alternative (IB) conformation of U48 with a blob of density in the original U48 position corresponding to the adenine ligand. The alternative conformation of A21 was much less pronounced and is partially disordered along with the adjacent hinge residues (U22 and A23). c, Keeping the occupancy of A21 at 0.5, the structure was refined with U48 omitted. The Fo − Fc map (3σ, green) again supports the alternative configuration of U48, A21, and density for the adenine ligand. d, The final refined structure of the IB state with the adenine ligand (red), and alternative conformations for both U48 and A21 modelled at 0.5 occupancy, shown with 2 Fo − Fc electron density map (1σ, blue).

Extended Data Figure 7 Fo − Fc and 2 Fo − Fc electron density maps, and ensemble refinement model for the 10-s-mix data.

a, Weighted Fo − Fc difference electron density maps in green (3σ) and red (−3σ), computed using the apo structure (apo2, cyan; apo1, blue) and the structure factors from the 10-s-mix data. The isolated peaks, most likely corresponding to backbone phosphates, indicate a mixture of conformational states, and are predominantly in and around the three-way junction of the apo2 structure. b, 2 Fo − Fc electron density map (1σ, left) and time-averaged molecular dynamics ensemble refinement model (right) for the ‘apo1-like’ molecule of the 10-s-mix structure. c, Superimposition of apo1 (blue) and the apo1-like molecule of the 10-s-mix structure (orange), indicating no structural changes to apo1 after 10 s of mixing with ligand.

Extended Data Table 1 Crystal data and refinement statistics
Full size table
Extended Data Table 2 R.m.s.d. values of all atoms among apo and ligand-bound conformers
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Extended Data Table 3 Average B-factors (Å2)
Full size table

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Stagno, J., Liu, Y., Bhandari, Y. et al. Structures of riboswitch RNA reaction states by mix-and-inject XFEL serial crystallography. Nature 541, 242–246 (2017). https://doi.org/10.1038/nature20599

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