Recognition of the bacterial second messenger cyclic diguanylate by its cognate riboswitch

Abstract

The cyclic diguanylate (bis-(3'-5')-cyclic dimeric guanosine monophosphate, c-di-GMP) riboswitch is the first known example of a gene-regulatory RNA that binds a second messenger. c-di-GMP is widely used by bacteria to regulate processes ranging from biofilm formation to the expression of virulence genes. The cocrystal structure of the c-di-GMP responsive GEMM riboswitch upstream of the tfoX gene of Vibrio cholerae reveals the second messenger binding the RNA at a three-helix junction. The two-fold symmetric second messenger is recognized asymmetrically by the monomeric riboswitch using canonical and noncanonical base-pairing as well as intercalation. These interactions explain how the RNA discriminates against cyclic diadenylate (c-di-AMP), a putative bacterial second messenger. Small-angle X-ray scattering and biochemical analyses indicate that the RNA undergoes compaction and large-scale structural rearrangement in response to ligand binding, consistent with organization of the core three-helix junction of the riboswitch concomitant with binding of c-di-GMP.

Figure 1

Overall structure of the c-di-GMP riboswitch. (a) Unbiased |F_o|-|F_c| electron density corresponding to the bound c-di-GMP before it was included in the crystallographic model, superimposed on the refined model of the ligand (map contoured at 3.0 s.d.) (b) Portion of a composite simulated annealing-omit 2|F_o|-|F_c| Fourier synthesis calculated with the final crystallographic model, contoured around A82 at 1.5 s.d. (c) Schematic representation of the structure of the riboswitch bound to c-di-GMP. Thin black lines with arrowheads depict connectivity, dashed green lines, long-range stacking interactions, and the red line the inter-helical base pair. Leontis-Westhof symbols depict non-canonical base pairs. Except for the U1A binding site, the numbering scheme is that of ref. , which is used throughout. (d) Cartoon representation of the three-dimensional structure. Color coding as in (c). The U1A-RBD is shown as a gray ribbon.

Figure 2

Specific binding of c-di-GMP. (a) The J1b/2 region stitches the three-helix junction and provides a nucleotide that intercalates between the c-di-GMP. View from the direction of Fig. 1d. Note alternation of J1b/2 residues between right (P1a/P1b) and left (P2). (b) View of the binding site from the major groove. (c) View from the minor groove. Additional interactions between the Watson-Crick faces of G19 and A47, and the sugar edge of A49 and g_II are suggested by the location of hydrogen-bond donors and acceptors (colored blue or red denoting nitrogen or oxygen, respectively), but remain uncertain at the current resolution limit. Dashed lines denote putative hydrogen bonds (distance cutoff=3.8 Å; note that the mean precision of the atomic coordinates is 0.5 Å). In panel (c), the hydrogen bond marked (*) is between the N6 of A18 and the N3 of g_I, not between G19 and the c-di-GMP.

Figure 3

C-di-GMP and Mg²⁺ induced size and shape changes of the riboswitch, monitored by SAXS. Red indicates 2.5 mM Mg²⁺; black 10 mM Mg²⁺. Dots denote c-di-GMP-free conditions; solid lines, c-di-GMP-bound conditions ("a.u.", arbitrary units). (a) Kratky plot suggests local disorder of the ligand-free riboswitch even under high MgCl₂ concentration (black dots). (b) Electron-pair probability analysis reveals compaction of the riboswitch upon binding the second messenger.

Figure 4

Global rearrangement of the riboswitch induced by c-di-GMP binding. (a) Low-resolution molecular envelope reconstruction based on SAXS data of the riboswitch in the presence of c-di-GMP (at 2.5 mM Mg²⁺). The maximum linear dimension of the reconstruction is ~90 Å. (b) Molecular envelope based on SAXS data of the riboswitch in the absence of c-di-GMP (at 2.5 mM Mg²⁺). The maximum linear dimension of the reconstruction is ~100 Å. The two arms of this reconstruction have dimensions approximately corresponding to those of P2 and P1b. (c) Results of nuclease probing summarized on the bound-state secondary structure. Stars denote RNase T1 protection upon c-di-GMP binding; filled ovals, RNase V1 protection upon c-di-GMP binding; open ovals, increased RNase V1 cleavage upon c-di-GMP binding. (d) Section of the autoradiogram highlighting protections observed due to their presence in the inter-helical interface, or indirectly, from stabilization of the interface between P1b and P2. (See Supplementary Fig. 10 for complete autoradiogram; this panel shows the lanes corresponding to 3.0 mM Mg²⁺). "+" and "−" denote conditions with 70 µM and 0 µM c-di-GMP, respectively. "NR" and "BH" denote "no reaction" and "base hydrolysis".