Structural basis for Diels-Alder ribozyme-catalyzed carbon-carbon bond formation

Abstract

The majority of structural efforts addressing RNA's catalytic function have focused on natural ribozymes, which catalyze phosphodiester transfer reactions. By contrast, little is known about how RNA catalyzes other types of chemical reactions. We report here the crystal structures of a ribozyme that catalyzes enantioselective carbon-carbon bond formation by the Diels-Alder reaction in the unbound state and in complex with a reaction product. The RNA adopts a lambda-shaped nested pseudoknot architecture whose preformed hydrophobic pocket is precisely complementary in shape to the reaction product. RNA folding and product binding are dictated by extensive stacking and hydrogen bonding, whereas stereoselection is governed by the shape of the catalytic pocket. Catalysis is apparently achieved by a combination of proximity, complementarity and electronic effects. We observe structural parallels in the independently evolved catalytic pocket architectures for ribozyme- and antibody-catalyzed Diels-Alder carbon-carbon bond-forming reactions.

Figure 1

Secondary and tertiary folds of the Diels-Alder ribozyme. (a) Ribozyme secondary structure (left) with stems I–III in cyan, gold and green, respectively, and residues found invariant in the selection procedure in red. The same colors are used throughout this figure. The ribozyme consists of a 11-mer (G1–C11) HEG-linked with anthracene and a 38-mer (G12–C49). The dashed line indicates a loop present in the selected ribozyme and deleted in the bipartite system. The ribozyme-catalyzed Diels-Alder reaction between anthracene and N-pentyl maleimide results in formation of a single enantiomer of the complex (right). (b,c) Tertiary fold (b) and three-dimensional topology (c) in the crystal structure of the ribozyme–product complex. The RNA bases and bound product (blue) are cylinders in c. The backbone is depicted with a ribbon, whereas the hydrated Mg²⁺ cations are in a mesh representation.

Figure 2

Details of the structure of the Diels-Alder ribozyme–product complex. (a) Final 2F_o – F_c electron density map (green), contoured at 1 σ, for the cycloaddition product and surrounding RNA. The product (blue) and RNA (red), superimposed with the density, are from the refined model. (b) RNA tertiary structure surrounding the ribozyme catalytic pocket containing bound product. The product is blue, adjacent A3-U45 and U23•A43 base pairs and U42•(G2-C25) base triple are light red, G24 is green, with additional layers moving farther out from the pocket in gold (A41•(C26-G2), G22-C46 and C44-G4), light green (A40•G27, C21-G47 and G18-C5), cyan (C20-G48) and orange (G19-C49). The hydrated Mg²⁺ cations are in magenta-colored mesh. (c) Tertiary structure in the region of stems I and II. The RNA is slightly tilted compared with b to highlight the relative alignment of the stems. Individual base pairs and hydrated Mg²⁺ cations are shown as in b.

Figure 3

Structural elements of the Diels-Alder ribozyme–product complex. The view is rotated 180° along the vertical axis relative to the schematic in Figure 1b. The color code is the same as in Figure 2, unless specified otherwise. Oxygen, nitrogen and phosphorus atoms are, respectively, red, blue and yellow balls. Hydrogen bonds are dotted lines. Coordination bonds to Mg atom are magenta sticks. (a) RNA segment aligned above the product. The A43 backbone is silver. (b) RNA segment aligned below the product. (c) Structural motif highlighting splaying out of G18 and G19.

Figure 4

Surface representation of the catalytic pocket. (a,b) Front (a) and back (b) views of the catalytic pocket of the ribozyme–product complex. (c) Front view of the free ribozyme. Mg²⁺ ions are shown as hydrated cations in a and b for the 3.0-Å structure, and as balls in c for the 3.5-Å structure.

Figure 5

Superposition of the Diels-Alder ribozyme structures around the catalytic pocket. The RNA structures in stick representation are from the ribozyme–product complex (red) and the free ribozyme (blue).

Figure 6

Comparison of the RNA and protein catalytic sites for the Diels-Alder reaction. The surface of the bound ligand is in mesh representation. (a) Catalytic pocket from the Diels-Alder ribozyme–product complex. (b) Catalytic pocket of the antibody catalyzing the retro-Diels-Alder reaction (PDB entry 1LO2). Anchoring of the ligand is achieved through direct and water-mediated (W127) hydrogen bonds.