The structure of the SAM/SAH-binding riboswitch

Abstract

S-adenosylmethionine (SAM) is a central metabolite since it is used as a methyl group donor in many different biochemical reactions. Many bacteria control intracellular SAM concentrations using riboswitch-based mechanisms. A number of structurally different riboswitch families specifically bind to SAM and mainly regulate the transcription or the translation of SAM-biosynthetic enzymes. In addition, a highly specific riboswitch class recognizes S-adenosylhomocysteine (SAH)-the product of SAM-dependent methyl group transfer reactions-and regulates enzymes responsible for SAH hydrolysis. High-resolution structures are available for many of these riboswitch classes and illustrate how they discriminate between the two structurally similar ligands SAM and SAH. The so-called SAM/SAH riboswitch class binds both ligands with similar affinities and is structurally not yet characterized. Here, we present a high-resolution nuclear magnetic resonance structure of a member of the SAM/SAH-riboswitch class in complex with SAH. Ligand binding induces pseudoknot formation and sequestration of the ribosome binding site. Thus, the SAM/SAH-riboswitches are translational 'OFF'-switches. Our results establish a structural basis for the unusual bispecificity of this riboswitch class. In conjunction with genomic data our structure suggests that the SAM/SAH-riboswitches might be an evolutionary late invention and not a remnant of a primordial RNA-world as suggested for other riboswitches.

Figure 1.

The SAM/SAH riboswitch and its ligands. (A) Chemical structures of SAM (top) and SAH (bottom). (B) Consensus sequence and secondary structure of the SAM/SAH-binding riboswitches. The ribosomal binding site is highlighted in bold letters. (C) Secondary structure of the SAM/SAH-binding riboswitch variant env9b and the P1 stem mutant env9bP1. The ribosomal binding site is highlighted in bold letters. The pseudoknot interactions indicated by connecting lines in (B) and (C) conform to the structure determined in this work and differ slightly from the original prediction. (D) Imino proton spectra of the env9bP1 RNA in the absence (top) and the presence of an 1.5-fold excess of SAH (middle) and 2 mM magnesium acetate (bottom). Resonance assignments in the SAH and Mg²⁺ bound state are indicated.

Figure 2.

The env9 SAM/SAH-riboswitch binds SAM and SAH with a similar binding mode and with similar affinities. (A) Overlay of the ¹⁵N-HSQC spectra of the imino region of uniformly ¹⁵N-labeled env9bP1 RNA in complex with SAH (black) and SAM (red) recorded at 20°C in the presence of 2 mM magnesium acetate. NMR signal assignments are indicated. (B) Representative ITC thermograms for the env9bP1 riboswitch titrated with SAH (left) or SAM (right) in the presence of 2 mM magnesium acetate. The resulting K_D values are indicated.

Figure 3.

Overall structure of the SAM/SAH-riboswitch in complex with SAH. (A) Superimposition of the ten riboswitch/SAH complex structures with the lowest CYANA target function. The base triplet between the bases G9, C14 and G39 is shown in dark blue, U16 and SAH in magenta, the base triplet C8:G17:U36 in green, the base triplet of A6:A18:A35 in light blue and A6 and C19 in orange. (B) Average structure of the riboswitch/SAH complex with the same coloring as used in (A). (C) Surface representation of the riboswitch RNA and SAH in stick representation with the same coloring as in (A). (D) Structure diagram of the complex structure using the Leontis-Westhof notation. (E) {¹H},¹³C hetNOE values for the aliphatic H1′/C1′- (solid black lines) and aromatic H8/C8- and H6/C6- (dashed gray lines) moieties along the sequence of the riboswitch in the SAH-bound state. The average hetNOE-values of the helical residues for aliphatic H1′/C1′ aromatic H6/C6 and H8/C8 groups are marked by a solid and a dashed red line, respectively. The different structural regions of the riboswitch are indicated by different background colors. (F) Imino proton spectra of the mutant with a truncated linker env9bΔ28-33 in the ligand-free state (top) and in the presence of 3-fold excess of SAH and 7.5 mM magnesium acetate (bottom) at 10°C.

Figure 4.

The SAH binding site. (A) HNN-COSY experiment on a ¹⁵N-U-labeled RNA in complex with ¹³C¹⁵N-labeled SAH showing a correlation between the U16 imino group and the N7 nitrogen of SAH. (B) SAH recognition in a ‘reversed Hoogsteen’ (trans Hoogsteen/Watson–Crick) base pair between SAH and U16 in a bundle representation. Hydrogen bonds are indicated by dashed lines. (C) Interactions between the sulfur atom and the ribose moiety of SAH with the C15:G38 base pair forming the ‘roof’ of the SAH-binding site. Dashed gray lines indicate hydrogen bonds and a dashed red line indicates the putative chalcogen bond between U16 and SAH. (D) The C8:G17:U36 minor groove base triplet (carbon atoms in green) forming the floor of the SAH binding pocket in a bundle representation. (E) Stacking interactions between the U16:SAH intermolecular base pair (carbon atoms in magenta) and the C8:G17:U36 (carbon atoms in green) minor groove base triplet. Hydrogen bonds are indicated by dashed lines. A black arrow points toward the O4′ oxygen of the SAH ribose group located atop the base of C8 in an orientation suitable for a lone pair–π interaction.

Figure 5.

Additional tertiary interactions in the vicinity of the ligand binding pocket. (A) Tertiary structure elements surrounding the intermolecular U16:SAH base pair (magenta). The Watson–Crick base pair C15:G38 at the base of stem P2 is shown in white and the base triplet C8:G17:U36 closing P1 is shown in green. Directly underneath this base triplet is the A7:A18:A35 base triplet (light blue) and the A6:C29 base pair (orange). A major groove base triplet G9:C14:G39 (dark blue) is located directly above the P2 closing base pair C15:G38 (white). (B) The G9:C14:G39 base triplet (dark blue) in a bundle representation. (C) Strip from the ¹H,¹H-NOESY at the H1 G9 resonance showing NOE contacts to neighboring nucleotides. (D) Imino proton spectra of the mutant env9bP1C14G/G39C without (top) and with 1.5-fold excess of SAH and 2 mM magnesium acetate (bottom). In the mutant with the inverted G:C base pairing orientation the base triplet with G9 cannot form and ligand binding is abrogated.

Figure 6.

The adenine base triplet A7:A18:A35. (A) Structure diagram of the complex structure using the Leontis–Westhof notation. The base triplet A7:A18:A35 is colored in light blue. (B) The A7:A18:A35 base triplet in a bundle representation. (C) Strip from the ¹H,¹H-NOESY-spectrum at the H2/H8 A35 resonances showing diagnostic NOE contacts between the A35 base protons and neighboring nucleotides. Signal assignments are indicated. (D) Overlay of the ¹⁵N-HSQC spectra of the imino region of uniformly ¹⁵N-labeled env9b RNA (black) and env9bA18G (light blue) in complex with SAH recorded at 20°C. The inset shows the geometry of the base triplet upon A18 replacement with G. (E) Representative ITC thermograms for env9bA18G titrated with SAH (left) or SAM (right). The resulting K_D values are indicated.

Figure 7.

The non-canonical A6:C19 base pair. (A) Structure diagram of the complex structure using the Leontis–Westhof notation. The non-canonical base pair A6:C19 is colored in orange. (B) The A6:C19 base pair in a bundle representation. (C) HNN-COSY experiment for the identification of the hydrogen bond between the N4 amino group of C19 and the N3 nitrogen of A6. In this experiment the amino group protons of C19 simultaneously show cross peaks to the N3 nitrogen of C19 due to the intranucleotide through-bond ²J_N4(C)N3(C) scalar coupling as well as to the A6 N3 nitrogen due to the internucleotide cross-hydrogen-bond ^2hJ_N4(C)N3(A) scalar coupling. (D) Strips from the ¹H,¹³C-NOESY-HSQC at the H2 A6 and H5 C19 resonance frequencies showing the strong NOEs between the H5 proton of C19 and the H2 proton of A6 supporting the unusual geometry of this A:C base pair. (E) Representative ITC thermogram for the titration of the env9bP1A6G riboswitch mutant with SAM. In this mutant a Watson–Crick G6:C19 base pair would replace the A:C base pair of the WT. The resulting K_D value is indicated.