Crystal structure of an RNA aptamer bound to thrombin

Abstract

Aptamers, an emerging class of therapeutics, are DNA or RNA molecules that are selected to bind molecular targets that range from small organic compounds to large proteins. All of the determined structures of aptamers in complex with small molecule targets show that aptamers cage such ligands. In structures of aptamers in complex with proteins that naturally bind nucleic acid, the aptamers occupy the nucleic acid binding site and often mimic the natural interactions. Here we present a crystal structure of an RNA aptamer bound to human thrombin, a protein that does not naturally bind nucleic acid, at 1.9 A resolution. The aptamer, which adheres to thrombin at the binding site for heparin, presents an extended molecular surface that is complementary to the protein. Protein recognition involves the stacking of single-stranded adenine bases at the core of the tertiary fold with arginine side chains. These results exemplify how RNA aptamers can fold into intricate conformations that allow them to interact closely with extended surfaces on non-RNA binding proteins.

FIGURE 1.

Secondary structure of the aptamer and detailed interactions with the protein. (A) Secondary structure of the aptamer. Base pairs are indicated by filled circles or a dashed line. Mutations and their effect on affinity are indicated in gray (White et al. 2001). (B) Interactions with the protein. Interactions are colored according to type: red, ion pair; green, hydrogen bond; brown, van der Waals contact; blue, stacking interaction between protein and RNA. In the RNA diagram, rectangles represent bases, pentagons represent sugars, open circles represent the backbone phosphate position, and closed circles represent the 2′ position of the sugar ring.

FIGURE 2.

Overall structure of the aptamer–thrombin complex. (A) Human thrombin (gray) complexed with the RNA aptamer (green), in stereo. Base-paired nucleotides are shown as yellow sticks, while single-stranded nucleotides are blue. Two protein arginine residues (brown) stack with single-stranded adenine bases. The protease inhibitor PPACK (dark red) is shown in the catalytic active site of thrombin. (B) The A-Arg zipper, shown as a stereo representation. Two protein arginines (yellow) intercalate with adenine RNA bases (green).

FIGURE 3.

Surface and electrostatic complementarity at the protein–RNA interface. (A) Overall structure of the complex is shown with the molecular surface of thrombin colored according to surface electrostatic potential (red, negative; blue, positive) and the RNA aptamer as green and yellow sticks. (B) Complementary surfaces of protein and RNA. Here, the RNA molecule has been detached from the protein and rotated 180° to reveal the molecular surfaces buried in the complex (colored yellow and green). A series of protrusions and corresponding depressions are labeled with the letters A–F, with uppercase and lowercase type indicating protrusions and depressions, respectively. The primary residue responsible for each protrusion is indicated in the key. Buried surfaces were calculated using the SC algorithm (Lawrence and Colman 1993) with water molecules excluded and illustrated in GRASP (Nicholls 1992). (C) Side view of the interacting surfaces. The complex has been rotated 90° to the right from the orientation in A. The surface illustrated here has been drawn at positions where the molecular surface of the RNA (blue sticks) is <0.2 Å from the molecular surface of the protein (yellow sticks). The surface is colored magenta for van der Waals contacts and cyan for hydrogen bonds. The lettering scheme is the same as in B.