Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch - PubMed (original) (raw)

Structural basis for specific, high-affinity tetracycline binding by an in vitro evolved aptamer and artificial riboswitch

Hong Xiao et al. Chem Biol. 2008.

Abstract

The tetracycline aptamer is an in vitro selected RNA that binds to the antibiotic with the highest known affinity of an artificial RNA for a small molecule (Kd approximately 0.8 nM). It is one of few aptamers known to be capable of modulating gene expression in vivo. The 2.2 A resolution cocrystal structure of the aptamer reveals a pseudoknot-like fold formed by tertiary interactions between an 11 nucleotide loop and the minor groove of an irregular helix. Tetracycline binds within this interface as a magnesium ion chelate. The structure, together with previous biochemical and biophysical data, indicates that the aptamer undergoes localized folding concomitant with tetracycline binding. The three-helix junction, h-shaped architecture of this artificial RNA is more complex than those of most aptamers and is reminiscent of the structures of some natural riboswitches.

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Figures

Figure 1

Figure 1. Overall structure of the tetracycline aptamer

A, Sequence of a wild-type connectivity tetracycline aptamer. Residue numbers correspond to those of the constructs of Hanson et al. (2005) and Müller et al. (2006). Gray nucleotides are functionally dispensable. B, Sequence of the circularly-permuted crystallization construct. Note deletion of loop L2, and introduction of an U1A protein binding site capping paired region P1. C, Chemical structure of tetracycline, numbered conventionally. X is hydrogen or chlorine, for tetracycline and 7-chlorotetracycline, respectively. D, Schematic secondary structure of the tetracycline-bound conformation of the aptamer. Nucleotides that form the antibiotic binding site are boxed in green. Thin lines with embedded arrowheads depict connectivity. Watson-Crick and single-hydrogen bond pairs are indicated by thin and dashed lines, respectively. Non-canonical base pair symbols are those of Leontis and Westhof (2001). Asterisk indicates that the base of G57 makes single hydrogen bonds with the bases of both, C10 and A11. E, Cartoon representation of the tetracycline aptamer (color-coded as in panel D) in complex with 7-chlorotetracycline (green stick figure) and U1A. Selected well-ordered magnesium ions and their solvation waters (magenta and red spheres, respectively) are shown. Colored arrows denote chain direction.

Figure 2

Figure 2. Structure of the aptamer core (color-coding as in Figure 1)

A, Interaction of L3 with the minor groove of the irregular helix formed by J1/2 and J2/3. Two-headed arrows denote selected hydrogen bonds. B, Structure of the core triplex. View is approximately perpendicular to that of panel A. C, Detail of the top of L3. A well-ordered cation with two crystallographically resolved coordination waters and a water molecule bridging N3 of A52 and 2′-OH of U54 are shown. View is approximately that of panel A.

Figure 3

Figure 3. Structure of the tetracycline binding site

A, Solvent-accessible surface of the aptamer colored by curvature (green and grey are convex and concave, respectively) with a molecular surface representation of the bound 7-chlorotetracycline. In this panel only, carbon atoms of the antibiotic are drawn black. Inset: ball-and-stick figure of the antibiotic and its coordinated, hydrated magnesium ion in the same orientation as in the surface representation. Note complete occlusion of the hydroxyl group at position 6β in the aptamer complex. B, Detail of the binding site. C, view of the binding site from the direction of L3, superimposed on the unbiased residual |_F_o|− |_F_c| electron density into which the antibiotic-magnesium complex was built, contoured at 3 and 5 s.d. (gray and red mesh, respectively).

Figure 4

Figure 4. Water molecules and hydrated magnesium ions in the aptamer-tetracycline complex

A, Well-ordered water molecules fill the major groove of L3 linking the exterior of the complex with the α face of the antibiotic. View is from the rear of Figure 1D. B, Magnesium ions line the junction between the major groove of P3 and the backbones of J1/2 and J2/3. Asterisks in panels A and B denote the same magnesium ion. Although all three magnesium ions have octahedral coordination geometry, only the crystallographically resolved ligands are shown.

Figure 5

Figure 5. Biochemical evidence for tetracycline-binding induced aptamer reorganization

A, Results of chemical probing of the unliganded aptamer by Hanson et al. (2005) plotted on the cocrystal structure. Dark, medium and light blue denote most strongly, intermediate, and most weakly modified nucleotides, respectively. B, Magnitude of the change in chemical modification between the unliganded and liganded tetracycline aptamer, determined by Hanson et al. (2005) plotted on the crystal structure. Nucleotides with the largest changes are denoted by deep purple, intermediate changes by magenta, and smallest by pink. C, Portion of the experimental electron density (Fourier synthesis calculated with the density-modified MAD phases) corresponding to the C10•C56 trans Watson-Crick pair contoured at 1 and 3 s.d. (blue and green mesh, respectively) superimposed on the final crystallographic model.

Figure 6

Figure 6. Comparison of ribosomal and protein tetracycline binding sites

A, Detail of the primary tetracycline binding site in the 3.4 Å-resolution cocrystal structure of the 30S ribosomal subunit of Thermus thermophilus (Brodersen et al., 2000). B, Detail of the ligand binding site of the 2.1 Å-resolution structure of the complex between the class D tetracycline repressor protein (TetR) and 7-chlorotetracycline (Hinrichs et al., 1994; Kisker et al., 1995). The binding site is formed by amino acid residues from both protomers of the dimeric protein. Amino acid residues from the second protomer are colored magenta.

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