Real-time crystallographic studies of the adenine riboswitch using an X-ray free-electron laser

Abstract

Structures of the four reaction states of the adenine riboswitch aptamer domain, including a transient intermediate state were solved by serial femtosecond crystallography. The structures not only demonstrate the use of X-ray free-electron lasers for RNA crystallography but have also proven that transient states can be determined in real time by mix-and-inject crystallography. These results illustrate the structural basis for the ligand-induced conformational changes associated with the molecular 'switch'.

Keywords: RNA; X-ray free-electron laser; adenine riboswitch; diffusive mixing; lattice conversion; phase transition; reaction intermediate; serial femtosecond crystallography; time-resolved crystallography.

Figure 1.

(A) Secondary structure of the V. vulnificus add adenine riboswitch and (B) Crystal structure of the ligand-free aptamer domain in apo1 (left, blue) and apo2 (right, cyan) states (PDB:5E54), aligned to the ligand-bound structure (grey, PDB:4TZX) for comparison. The P1 helix (yellow), J1/2 hinge (red), and J2/3 latch (green) regions, which exhibit the greatest structural differences among rA71 conformational states, are highlighted. The switching sequence is denoted in red bold letters, the Shine-Dalgarno in orange, and the start codon in purple. The sequence used for the apo-rA71 structure differs slightly from what is shown in (A) with a few stabilizing mutations in P1.

Figure 2.

(A-B) Two-dimensional projections (XY-plane) of the structural coordinates (Å) of key residues for apo2 and B•ade. The structures of apo2 (PDB:5E54) and B•ade (PDB:4TZX) were aligned as in Fig. 1, and the XY-coordinates for O2 atoms (pyrimidines) or N6 atoms (purines) were taken directly from their respective PDB files and plotted. (A) “Swinging residues” that flip toward (blue) or away (red) from the ligand-binding pocket upon conversion to B•ade. The direction of movement is also indicated by dotted lines. Values reported above the dotted lines correspond to the distances in three dimensions. (B) Residues involved in the three ligand-facilitated base triples: U20-U49-A76 (yellow), A21-C50-U75 (grey), and U47-U51-U74-ade (green). (C) In the absence of ligand (apo1 and apo2), the base-triple interactions are broken. Ligand binding facilitates the formation of the base triples, as observed in the crystal structure of ligand-bound rA71 (PDB:4TZX). These interactions include ligand recognition by U74 and anchoring of P1 (residues U75 and A76) and the hinge (U20 and A21) via the latch (residues U47, U49, C50 and U51), ultimately leading to the stabilization of the P1 helix.

Figure 3.

Large conformational changes induced after 10 minutes of ligand mixing result in a polymorphic phase transition and lattice conversion from a monoclinic (apo1 and apo2: blue and cyan respectively) to an orthorhombic space group (B•ade: magenta), which was accommodated in micro/nanocrystals of rA71. A subset of 4 related molecules (white) between the two structures illustrates that half of the molecules rotate ~90° upon conversion.