Opening Image: Ternary complex of the antibiotic thiostrepton (CPK), a 58-nucleotide fragment of 23S rRNA, and the ribosomal protein L11. Toggle spacefill.

Outline

1. Introduction
2. Protein L11
   1. 70S Bacterial Ribosome
   2. Terminal Domains
   3. rRNA Recognition
   4. In Motion
3. Tertiary Structure of L11-Binding Domain
   1. Ribose Zipper
   2. Non-Polar Core
   3. Stabilization by L11
4. Cooperative Binding of Thiostrepton
5. Summary

Introduction

Protein-nucleic acid interactions

Many fundamental processes in biology are governed by protein-nucleic acid interactions. Protein-nucleic acid interfaces contain a variety of noncovalent interactions, including hydrogen bonding, charge-charge interactions, cation-π and π-π interactions, water hydrogen-bond bridging, and hydrophobic interactions. Protein-DNA interactions generally involve a short stretch of double-helical DNA, which can sometimes be bent or kinked. Since noncoding RNA molecules fold into complex tertiary structures, recognition by proteins is inherently more complicated.

An Overview of the Ribosome

The ribosome is a large protein-RNA complex that catalyzes protein synthesis in all organisms. It consists of two subunits, which together contain over 50 different proteins in association with several large RNA molecules. In bacteria, the small and large subunits are designated 30S and 50S, respectively, on the basis of their sedimentation coefficient in Svedberg units (S). The structural scaffolding of the 30S subunit is 16S rRNA (≈1500 nucleotides (nt) in length); the 50S subunit contains 23S rRNA (≈2900 nt), 5S rRNA (≈120 nt), and more than 30 different proteins. Thus, the whole 70S ribosome contains about 4500 nucleotides and more than 50 various proteins.

The translation of genetic information encoded by the mRNA template into the protein polypeptide chain is executed by the ribosome. The most important functional sites are associated with highly conserved domains of ribosomal RNAs. The tRNA substrates are bound at the interface between the 30S small subunit and the 50S large subunit. In each of the three tRNA binding sites, the ribosome contacts all of the major elements of tRNA, providing an explanation for the conservation of tRNA structure. Ribosomal proteins are generally bound at the periphery (see model below). Synthesis of the peptide bond is catalyzed by 23S rRNA. Two GTP molecules are hydrolyzed for each amino acid residue that is incorporated during elongation of the polypeptide chain. Their hydrolysis occurs at the GTPase center of the large subunit and drives a series of conformational changes in the ribosome.

Essential Points

This at-a-glance view of protein-RNA interactions introduces several important concepts:

• Many protein and RNA molecules have substructures called domains that can fold independently.
• The stability of a protein or RNA molecule depends not only on tertiary (intramolecular) interactions but also on its interactions with other ligands.
• Proteins are not rigid and can often adjust their conformations during association with ligands. This is the induced-fit mechanism.
• The binding of one ligand can facilitate the binding of another ligand. This synergism is called cooperative binding.

Protein L11

This tutorial focuses on interactions of the ribosomal protein L11 with its binding site on 23S rRNA. The L11 binding site is one of the most important functional sites in the ribosome. This domain, which is highly conserved in archea, bacteria, and eukarytic organisms, comprises nucleotides 1051 to 1109 (E. coli numbering) of 23S rRNA. Together with L11, it constitutes the GTPase center, one of the principle active sites of the large ribosomal subunit. The isolated 58-nucleotide domain folds independently into a unique molecular structure that is charmingly complex, but only marginally stable in the absence of L11. The antibiotic thiostrepton recognizes the same rRNA domain. Tight binding of this drug imposes conformational constraints on protein L11 and thereby inhibits protein synthesis.

70S Bacterial Ribosome

To understand ribosomal protein L11, we must first examine the 70S bacterial ribosome, shown here. The large and small subunits are shown in green and blue, respectively.

Isolate the large subunit. 23S and 5S rRNA are shown in dark and light green, respectively. The large ribosomal proteins are shown in violet.

Protein L11

Color the L11 protein and the L11 binding domain. The L11 binding domain and the ribosomal protein L11 form a knob at the outer edge of the large subunit.

N- and C-terminal Domains

L11 consists of two globular domains, a dynamic N-terminal domain (NTD) and the RNA-binding C-terminal domain (CTD), connected by a tripeptide linker.

Toggle backbone trace of L11. The N-terminal domain is colored blue (for the nitrogen atom of the N-terminal amino group), while the C-terminal domain is shown in red (for the oxygen in the C-terminal carboxyl group). The polypeptide chain wanders back and forth within each domain. Three amino acids connect the two domains.

Toggle color coding of the C_α atoms in the polypeptide chain corresponding to the temperature factor. The N-terminal domain is more dynamic than the C-terminal domain (reds are "hotter" than blues). Learn more.

Toggle spacefilled model. Protein domain structure is obvious in a backbone model, but less obvious in a spacefilled model since the domains often make extensive contacts with each other.

Protein L11

Recognition of rRNA by the C-Terminal Domain

The rRNA domain, shown as a cartoon model, and a spacefill model of L11. Only two residues of the NTD interact with rRNA.

The association constant for binding of L11 to the L11-binding fragment of 23S rRNA is nearly 10¹⁰ M^-1. The interaction is mainly one of shape complementarity involving van der Waals and charge-charge interactions, since the yeast L11-binding domain can replace the corresponding region within E. coli 23S rRNA despite 20 base differences.

Highlight the twenty-three amino acid residues of CTD which interact with rRNA.

Eight of the 23 are found in the RNA binding loop (or RBL). The RNA binding loop of CTD is shown in orange; C_α atoms of amino acids in the loop that interact directly with rRNA are shown in spacefill. We'll return to the RBL later.

Six other amino acids are located in a rigid α-helix that packs tightly into a grove of the rRNA domain.

Altogether, one third of the amino acid residues in CTD interact with rRNA.

Protein L11

L11 In Motion

Protein L11 changes conformation upon binding to RNA. Both the C-terminal and N-terminal domains undergo movements that are necessary for their function. In particular, thiostrepton blocks protein synthesis by preventing conformational changes in NTD that are necessary for its function.

A computer simulation illustrates this change, starting with the solution structure and ending with conformation of L11 in the bound state.

Animate the allosteric transition during binding, showing a superposition of 14 models.

Let's focus on CTD, since this is the domain that binds to rRNA. The RNA binding loop, RBL, is colored a brighter shade than the rest of the protein.

Superimpose of the free and bound conformations of CTD. Aside from the 180° flip in the RNA binding loop, the backbone of the C-terminal domain is essentially identical in the two conformations. CTD has a better binding energy to rRNA than would be the case if the protein were completely rigid. Why is that so?

Animate without superposition.

Tertiary Structure of the L11-Binding Domain

It's time to explore some highlights of the tertiary interactions present in the rRNA component. Designated as the L11-binding domain or L11-BD, this 58-nt domain is very compact as a result of extensive tertiary interactions along the entire length of the structure. Many of the residues in the rRNA domain are highly conserved, either because they are intimately involved in specifying tertiary structure or because they are critical for function. The interactions stabilizing the L11-binding domain include a ribose zipper, a nonpolar core containing stacked base triples, and intermolecular interactions with Mg²⁺, water, and L11.

Toggle between spacefill and trace models representing the tertiary structure. See Fig. 1 for color key.

As always, Mg²⁺ and water play key roles in RNA tertiary structure. Show solvent molecules.

Note the bulged-out nucleotide from helix C that is inserted into the internal loop of helix A.

Let's focus on the site. A1088 forms a universally conserved pair with U1060. Return to the spacefilling model and confirm that A1088 is a bulged-out base from helix C.

Tertiary Structure

Ribose Zipper

In music or architecture, a motif is theme or design that appears repeatedly. An RNA structural motif is a 3D structural design or specific tertiary interaction observed frequently among known RNA structures. The motif includes on a complete 3D description, including backbone conformation, all hydrogen-bonding and base-stacking interactions, and sequence preferences. In addition, an RNA structural motif may have bound waters or metals to support its conformation.

The crystal structures of several archeal and bacterial ribosomes has created a virtual avalanche of RNA structural motifs. One example is the ribose zipper. This important motif is characterized by hydrogen-bonding between 2′-hydroxyls from two consecutive ribose sugars in one chain with two consecutive ribose residues in a second chain. All ribose zippers are formed by antiparallel chains and show sequence conservation. There are 16 ribose zippers in 16S rRNA and 44 in 23S rRNA. The ribose zipper allows close approach of two backbone segments and thus is very important to the compact folding observed in large RNAs.

Tertiary Structure

Non-Polar Core of L11-Binding Domain

Another major feature of the tertiary structure of the L11-binding domain is the nonpolar core formed by a cluster of stacked bases. The T_m of the rRNA fragment (41.5 °C) corresponds to melting of the nonpolar core; Which is to say, it's the "glue" that holds the different pieces of secondary structure together.

Identify the three base triples in this core.

Toggle spacefilling model. The nonpolar core is shown in CPK colors.

L11 Stabilizes the L11-Binding Domain

At saturating concentrations of L11, no unfolding of the RNA is seen at 40 °C; by stabilizing the set of tertiary interactions, the entire RNA structure is prevented from unfolding.

Spacefilling model of the binary L11 • rRNA complex. Protein L11 is colored violet. The association constant for binding of L11 to the L11-binding fragment of 23S rRNA is nearly 10¹⁰ M^-1 (ΔG° = -56.8 kJ/mol for the binding affinity of L11 at 37 °C). The L11 C-terminal domain is all that is needed to stabilize the RNA. Explain.

Cooperative Binding of Thiostrepton

Thiostrepton binds to the cleft between the RNA and N-terminal domain of L11. Thiostrepton is the only antibiotic drug to bind to both rRNA and a protein in the ribosome. Its binding site on 23S rRNA involves A1067 and A1095. Protein L11 is required for high-affinity binding of thiostrepton to the RNA. Thiostrepton has a much weaker affinity for the RNA alone (K_d = 0.4 μM), and it does not bind to L11 in solution.

The L11 • rRNA • thiostrepton ternary complex is shown in spacefill. The C-terminal domain of L11 is red and the N-terminal domain is blue. rRNA is shown in light green, while thiostrepton is shown in CPK colors.

The antibiotic binds in the gap between the RNA and the N-terminal domain of L11. Highlight the van der Waals contacts.

The ternary L11 • thiostrepton • RNA complex is 2300 times more stable than expected on the basis of L11 • RNA and thiostrepton • RNA binding affinities (see Fig. 2).

Summary

1. Ribosomal protein L11 and its associated binding site on 23S rRNA together comprise one of the principle active sites of the prokaryotic ribosome.
2. The L11-binding domain of the rRNA of the large ribosomal subunit is compactly folded into a well defined tertiary structure, which is further stabilized by the association with the C-terminal domain of L11.
3. Relatively large surfaces are typically involved in protein-RNA interactions.
4. The stability and specificity of protein-RNA complexes depend on the alignment of attractive noncovalent interactions in a complementary manner.
5. The L11 N-terminal domain mediates the cooperative binding of thiostrepton to rRNA. Thiostrepton binding compromises the ability of the N-terminal domain to interact with other components of the protein synthesizing machinery. It does so mainly by restricting the conformational flexibility of the N-terminal domain.