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

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 platform1–3. 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.

Microfluidics: From crystallization to serial time-resolved crystallography

“…29,30,35,220 By including an additional capillary into the GDVN architecture, diffusive mixing between the crystal slurry and an outer sheath of triggering solution can be achieved. The time for mixing can then be altered by adjusting the location of the mixing point within the larger GDVN structure.…”

“…141 This approach has been used to study the reaction of ß-lactamase microcrystals from M. tuberculosis with an antibiotic solution of ceftriaxone 29 and the binding of ligands to the adenine riboswitch adaptor domain. 30 For both these reports, crystals on the order of 1-10 lm in size were analyzed at time-delays on the order of 2-10 s. The use of the GDVN facilitated matching of the jet diameter to that of the crystals. While the mixing injector FIG.…”

“…[15][16][17][18] Furthermore, these approaches have enabled timeresolved structural determination for a much broader range of targets than had previously been accessible. [19][20][21][22][23][24][25][26][27][28][29][30] However, these large-scale serial methods suffer from the need to manipulate crystals and/ or from inefficient sample utilization. 3,[31][32][33][34][35][36] The material-intensive nature of these experiments extends the long-standing challenge of growing a well-diffracting crystal to reproducibly generating a large number of high quality, isomorphous crystals.…”

The Linac Coherent Light Source: Recent Developments and Future Plans

“…Recent LCLS time-resolved SFX studies of riboswitches, structural elements of messenger RNA (mRNA), capture the dynamic structural response to the binding of a ligand for the first time, as shown in Figure 6 [18]. This ligand-triggered conformational reaction in mRNA mediates gene expression.…”

Structure of the Ribosomal RNA Decoding Site Containing a Selenium‐Modified Responsive Fluorescent Ribonucleoside Probe

“…[26] Ther esponsiveness of the fluorophore to subtle conformational changes and the strong diffraction signal from the Se atom as demonstrated by using decoding site RNA suggest that such aprobe,when judiciously placed, would not only allow the concurrent investigation of RNAstructure and function but also could support integrated approaches to antibiotics discovery against resistant bacterial strains.…”