PDBsum entry 1f27

RNA

PDB id

1f27

Contents

			DNA/RNA

			Ligands

					BTN

			Metals

					_MG ×6

			Waters ×184

References listed in PDB file

Key reference

Title

The 1.3 a crystal structure of a biotin-Binding pseudoknot and the basis for RNA molecular recognition.

Authors

J.Nix, D.Sussman, C.Wilson.

Ref.

J Mol Biol, 2000, 296, 1235-1244. [DOI no: 10.1006/jmbi.2000.3539]

PubMed id

10698630

Abstract

A pseudoknot-containing aptamer isolated from a pool of random sequence molecules has been shown previously to represent an optimal RNA solution to the problem of binding biotin. The affinity of this RNA molecule is nonetheless orders of magnitude weaker than that of its highly evolved protein analogs, avidin and streptavidin. To understand the structural basis for biotin binding and to compare directly strategies for ligand recognition available to proteins and RNA molecules, we have determined the 1.3 A crystal structure of the aptamer complexed with its ligand. Biotin is bound at the interface between the pseudoknot's stacked helices in a pocket defined almost entirely by base-paired nucleotides. In comparison to the protein avidin, the aptamer packs more tightly around the biotin headgroup and makes fewer contacts with its fatty acid tail. Whereas biotin is deeply buried within the hydrophobic core in the avidin complex, the aptamer relies on a combination of hydrated magnesium ions and immobilized water molecules to surround its ligand. In addition to demonstrating fundamentally different approaches to molecular recognition by proteins and RNA, the structure provides general insight into the mechanisms by which RNA function is mediated by divalent metals.



	Figure 2. Figure 2. Specific interactions that stabilize the pseudoknot. (a) A stripe of magnesium ions lines the major groove of the stacked pseudoknot helices. Each magnesium ion (cyan) is coordinated by six ligands with square bipyramidal geometry. Water molecules (blue) complete the coordination shells of Mg1 and Mg2, and several directly hydrogen bond to major groove carbonyl group oxygen atoms and imidazole nitrogen atoms on the RNA. Coordination shells for Mg3 and Mg4 each include a single RNA oxygen atom serving as an axial ligand (the O4 carbonyl group of U7 and an A9 non-bridging phosphate oxygen atom, respectively) in addition to five water molecules. (b) Two stacked nucleotides, C8 and A9 (orange), connect the 5 -strands of the two helices to form loop 1. Mg4 and Mg5 are directly coordinated to phosphate oxygen atoms 30 and 5 to C8, helping to stabilize an unusual backbone conformation which inverts the nucleotide relative to those that flank it. Direct and water-mediated hydrogen bonds (broken lines) from the C8 base to helix 2 lock it in position and can account for its absolute conservation among biotin aptamers. (c) Two G-G stacks interact with magnesium ions. Mg2 interacts with G18 and G19, Mg4 with G12 and G13. In both examples hydrogen bonds are conserved with the N7 atom of the imidizole ring and carbonyl oxygen atoms of the base (with Mg4 recruiting an additional water molecule) and a water molecule is involved in positioning the metal ion with the backbone. (d) A23 is flipped out from the adenosine stack in loop 2 to pack across the minor groove of helix 1. Formation of a base triple with C4 delta G19 (similar to that in the BWYV pseudoknot; Su et al., 1999) and of a hydrogen bond chain linking the 20-hydroxyl groups of G19, A22, and A23 combine to stabilize the adenosine base. Figures prepared using RIBBONS (Carson, 1986) and Conic (Huang, 1991).		Figure 3. Figure 3. Biotin binding site. (a) The aromatic bases of A26 and G27 and the backbone connecting them interact tightly with the biotin thiophene ring. Packing with the A26 base and the U7 ribose moiety, direct coordination to a buried, solvated magnesium ion (Mg6), and hydrogen bonds to the G6 exocyclic amine and a frozen water molecule stabilize the ureido ring. (b) sA weighted 2Fo - Fc electron density map of a portion of the ligand binding pocket at 1.3 Å contoured at 1.0 s above the mean. Figures prepared using Conic (Huang, 1991) and RASTER3D (Merritt & Murphy, 1994).

	Headers