Gregers R. Andersen

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

An 11.7‐Å‐resolution cryo‐EM map of the yeast 80S·eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin–ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet‐like subunit rearrangement (RSR) occurs in the 80S·eEF2·sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.

Structure of the exon junction core complex with a trapped DEAD-box ATPase bound to RNA.

In higher eukaryotes, a multiprotein exon junction complex is deposited on spliced messenger RNAs. The complex is organized around a stable core, which serves as a binding platform for numerous factors that influence messenger RNA function. Here, we present the crystal structure of a tetrameric exon junction core complex containing the DEAD-box adenosine triphosphatase (ATPase) eukaryotic initiation factor 4AIII (eIF4AIII) bound to an ATP analog, MAGOH, Y14, a fragment of MLN51, and a polyuracil mRNA mimic. eIF4AIII interacts with the phosphate-ribose backbone of six consecutive nucleotides and prevents part of the bound RNA from being double stranded. The MAGOH and Y14 subunits lock eIF4AIII in a prehydrolysis state, and activation of the ATPase probably requires only modest conformational changes in eIF4AIII motif I.

Bacterial Polypeptide Release Factor RF2 Is Structurally Distinct from Eukaryotic eRF1

Bacterial release factor RF2 promotes termination of protein synthesis, specifically recognizing stop codons UAA or UGA. The crystal structure of Escherichia coli RF2 has been determined to a resolution of 1.8 A. RF2 is structurally distinct from its eukaryotic counterpart eRF1. The tripeptide SPF motif, thought to confer RF2 stop codon specificity, and the universally conserved GGQ motif, proposed to be involved with the peptidyl transferase center, are exposed in loops only 23 A apart, and the structure suggests that stop signal recognition is more complex than generally believed.

Structural Basis for Nucleotide Exchange and Competition with tRNA in the Yeast Elongation Factor Complex eEF1A:eEF1Bα

The crystal structure of a complex between the protein biosynthesis elongation factor eEF1A (formerly EF-1alpha) and the catalytic C terminus of its exchange factor, eEF1Balpha (formerly EF-1beta), was determined to 1.67 A resolution. One end of the nucleotide exchange factor is buried between the switch 1 and 2 regions of eEF1A and destroys the binding site for the Mg(2+) ion associated with the nucleotide. The second end of eEF1Balpha interacts with domain 2 of eEF1A in the region hypothesized to be involved in the binding of the CCA-aminoacyl end of the tRNA. The competition between eEF1Balpha and aminoacylated tRNA may be a central element in channeling the reactants in eukaryotic protein synthesis. The recognition of eEF1A by eEF1Balpha is very different from that observed in the prokaryotic EF-Tu:EF-Ts complex. Recognition of the switch 2 region in nucleotide exchange is, however, common to the elongation factor complexes and those of Ras:Sos and Arf1:Sec7.

Elongation factors in protein biosynthesis

Translation elongation factors are the workhorses of protein synthesis on the ribosome. They assist in elongating the nascent polypeptide chain by one amino acid at a time. The general biochemical outline of the translation elongation cycle is well preserved in all biological kingdoms. Recently, there has been structural insight into the effects of antibiotics on elongation. These structures provide a scaffold for understanding the biological function of elongation factors before high-resolution structures of such factors in complex with ribosomes are obtained. Very recent structures of the yeast translocation factor and its complex with the antifungal drug sordarin reveal an unexpected conformational flexibility that might be crucial to the mechanism of translocation.

Structures of modified eEF2.80S ribosome complexes reveal the role of GTP hydrolysis in translocation.

On the basis of kinetic data on ribosome protein synthesis, the mechanical energy for translocation of the mRNA–tRNA complex is thought to be provided by GTP hydrolysis of an elongation factor (eEF2 in eukaryotes, EF‐G in bacteria). We have obtained cryo‐EM reconstructions of eukaryotic ribosomes complexed with ADP‐ribosylated eEF2 (ADPR‐eEF2), before and after GTP hydrolysis, providing a structural basis for analyzing the GTPase‐coupled mechanism of translocation. Using the ADP‐ribosyl group as a distinct marker, we observe conformational changes of ADPR‐eEF2 that are due strictly to GTP hydrolysis. These movements are likely representative of native eEF2 motions in a physiological context and are sufficient to uncouple the mRNA–tRNA complex from two universally conserved bases in the ribosomal decoding center (A1492 and A1493 in Escherichia coli) during translocation. Interpretation of these data provides a detailed two‐step model of translocation that begins with the eEF2/EF‐G binding‐induced ratcheting motion of the small ribosomal subunit. GTP hydrolysis then uncouples the mRNA–tRNA complex from the decoding center so translocation of the mRNA–tRNA moiety may be completed by a head rotation of the small subunit.

Two crystal structures demonstrate large conformational changes in the eukaryotic ribosomal translocase

Two crystal structures of yeast translation elongation factor 2 (eEF2) were determined: the apo form at 2.9 Å resolution and eEF2 in the presence of the translocation inhibitor sordarin at 2.1 Å resolution. The overall conformation of apo eEF2 is similar to that of its prokaryotic homolog elongation factor G (EF-G) in complex with GDP. Upon sordarin binding, the three tRNA-mimicking C-terminal domains undergo substantial conformational changes, while the three N-terminal domains containing the nucleotide-binding site form an almost rigid unit. The conformation of eEF2 in complex with sordarin is entirely different from known conformations observed in crystal structures of EF-G or from cryo-EM studies of EF-G–70S complexes. The domain rearrangements induced by sordarin binding and the highly ordered drug-binding site observed in the eEF2–sordarin structure provide a high-resolution structural basis for the mechanism of sordarin inhibition. The two structures also emphasize the dynamic nature of the ribosomal translocase.

Structure of the haptoglobin–haemoglobin complex

Red cell haemoglobin is the fundamental oxygen-transporting molecule in blood, but also a potentially tissue-damaging compound owing to its highly reactive haem groups. During intravascular haemolysis, such as in malaria and haemoglobinopathies, haemoglobin is released into the plasma, where it is captured by the protective acute-phase protein haptoglobin. This leads to formation of the haptoglobin–haemoglobin complex, which represents a virtually irreversible non-covalent protein–protein interaction. Here we present the crystal structure of the dimeric porcine haptoglobin–haemoglobin complex determined at 2.9 Å resolution. This structure reveals that haptoglobin molecules dimerize through an unexpected β-strand swap between two complement control protein (CCP) domains, defining a new fusion CCP domain structure. The haptoglobin serine protease domain forms extensive interactions with both the α- and β-subunits of haemoglobin, explaining the tight binding between haptoglobin and haemoglobin. The haemoglobin-interacting region in the αβ dimer is highly overlapping with the interface between the two αβ dimers that constitute the native haemoglobin tetramer. Several haemoglobin residues prone to oxidative modification after exposure to haem-induced reactive oxygen species are buried in the haptoglobin–haemoglobin interface, thus showing a direct protective role of haptoglobin. The haptoglobin loop previously shown to be essential for binding of haptoglobin–haemoglobin to the macrophage scavenger receptor CD163 (ref. 3) protrudes from the surface of the distal end of the complex, adjacent to the associated haemoglobin α-subunit. Small-angle X-ray scattering measurements of human haptoglobin–haemoglobin bound to the ligand-binding fragment of CD163 confirm receptor binding in this area, and show that the rigid dimeric complex can bind two receptors. Such receptor cross-linkage may facilitate scavenging and explain the increased functional affinity of multimeric haptoglobin–haemoglobin for CD163 (ref. 4).

Structure of Eef3 and the Mechanism of Transfer RNA Release from the E-Site.

Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA–eEF1A–GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.

Crystal structures of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex.

In the elongation cycle of protein biosynthesis, the nucleotide exchange factor eEF1Bα catalyzes the exchange of GDP bound to the G-protein, eEF1A, for GTP. To obtain more information about the recently solved eEF1A–eEF1Bα structure, we determined the structures of the eEF1A–eEF1Bα–GDP–Mg2+, eEF1A–eEF1Bα–GDP and eEF1A–eEF1Bα–GDPNP complexes at 3.0, 2.4 and 2.05 Å resolution, respectively. Minor changes, specifically around the nucleotide binding site, in eEF1A and eEF1Bα are consistent with in vivo data. The base, sugar and α-phosphate bind as in other known nucleotide G-protein complexes, whereas the β- and γ-phosphates are disordered. A mutation of Lys 205 in eEF1Bα that inserts into the Mg2+ binding site of eEF1A is lethal. This together with the structures emphasizes the essential role of Mg2+ in nucleotide exchange in the eEF1A–eEF1Bα complex.