Morten Kjeldgaard

Crystal Structure of the Ternary Complex of Phe-tRNAPhe, EF-Tu, and a GTP Analog

The structure of the ternary complex consisting of yeast phenylalanyl-transfer RNA (Phe-tRNAPhe), Thermus aquaticus elongation factor Tu (EF-Tu), and the guanosine triphosphate (GTP) analog GDPNP was determined by x-ray crystallography at 2.7 angstrom resolution. The ternary complex participates in placing the amino acids in their correct order when messenger RNA is translated into a protein sequence on the ribosome. The EF-Tu-GDPNP component binds to one side of the acceptor helix of Phe-tRNAPhe involving all three domains of EF-Tu. Binding sites for the phenylalanylated CCA end and the phosphorylated 5′ end are located at domain interfaces, whereas the T stem interacts with the surface of the β-barrel domain 3. The binding involves many conserved residues in EF-Tu. The overall shape of the ternary complex is similar to that of the translocation factor, EF-G-GDP, and this suggests a novel mechanism involving “molecular mimicry” in the translational apparatus.

The crystal structure of elongation factor EF-Tu from Thermus aquaticus in the GTP conformation

BACKGROUND Elongation factor Tu (EF-Tu) is a GTP-binding protein that is crucial for protein biosynthesis. In the GTP form of the molecule, EF-Tu binds tightly to aminoacyl-tRNA, forming a ternary complex that interacts with the ribosomal acceptor site. During this interaction, GTP is hydrolyzed, and EF-Tu.GDP is ejected. RESULTS The crystal structure of EF-Tu from Thermus aquaticus, complexed to the GTP analogue GDPNP, has been determined at 2.5 A resolution and compared to the structure of Escherichia coli EF-Tu.GDP. During the transition from the GDP (inactive) to the GTP (active) form, domain 1, containing the GTP-binding site, undergoes internal conformational changes similar to those observed in ras-p21. In addition, a dramatic rearrangement of domains is observed, corresponding to a rotation of 90.8 degrees of domain 1 relative to domains 2 and 3. Residues that are affected in the binding of aminoacyl-tRNA are found in or near the cleft formed by the domain interface. CONCLUSION GTP binding by EF-Tu leads to dramatic conformational changes which expose the tRNA binding site. It appears that tRNA binding to EF-Tu induces a further conformational change, which may affect the GTPase activity.

Refined structure of elongation factor EF-Tu from Escherichia coli

The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution. In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles. The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels. The GDP-binding domain has a folding that is found in other GDP-binding proteins. Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.

The GTP binding motif: variations on a theme.

GTP binding proteins (G‐proteins) have wide‐ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP‐bound form and inactive in the GDP‐bound form. The protein synthesis elongation factor EF‐Tu was the first G‐protein whose nucleotide binding domain was solved structurally by X‐ray crystallography to yield a structural definition of the GDP‐bound form, but a still increasing number of new structures of G‐proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G‐proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF‐Tu and EF‐G, and the heterotrimeric G‐proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.—Kjeldgaard, M., Nyborg, J., Clark, B. F. C. The GTP binding motif: variations on a theme. FASEB J. 10, 1347‐1368 (1996)

Helix unwinding in the effector region of elongation factor EF-Tu–GDP

BACKGROUND Elongation factor Tu (EF-Tu) in its GTP conformation is a carrier of aminoacylated tRNAs (aa-tRNAs) to the ribosomal A site during protein biosynthesis. The ribosome triggers GTP hydrolysis, resulting in the dissociation of EF-Tu-GDP from the ribosome. The affinity of EF-Tu for other molecules involved in this process, some of which are unknown, is regulated by two regions (Switch I and Switch II) that have different conformations in the GTP and GDP forms. The structure of the GDP form of EF-Tu is known only as a trypsin-modified fragment, which lacks the Switch I, or effector, domain. The aim of this work was to establish the overall structure of intact EF-Tu-GDP, in particular the structure of the effector domain. RESULTS The crystal structures of intact EF-Tu-GDP from Thermus aquaticus and Escherichia coli have been determined at resolutions of 2.7 A and 3.8 A, respectively. The structures confirm the domain orientation previously found in the structure of partially trypsin-digested EF-Tu-GDP. The structures of the effector region in T. aquaticus and E. coli EF-Tu-GDP are very similar. The C-terminal part of the effector region of EF-Tu-GDP is a beta hairpin; in EF-Tu-GTP, this region forms an alpha helix. This conformational change is not a consequence of crystal packing. CONCLUSIONS EF-Tu undergoes major conformational changes upon GTP hydrolysis. Unlike other GTP-binding proteins, EF-Tu exhibits a dramatic conformational change in the effector region, involving an unwinding of a small helix and the formation of a beta hairpin structure. This change is presumably involved in triggering the release of tRNA, and EF-Tu, from the ribosome.

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.

NEW EMBO MEMBER’S REVIEWS: Macromolecular mimicry

Some proteins have been shown to mimic the overall shape and structure of nucleic acids. For some of the proteins involved in translating the genetic information into proteins on the ribosome particle, there are indications that such observations of macromolecular mimicry even extend to similarity in interaction with and function on the ribosome. A small number of structural results obtained outside the protein biosynthesis machinery could indicate that the concept of macromolecular mimicry between proteins and nucleic acids is more general. The implications for the function and evolution of protein biosynthesis are discussed.

Structural determination of the functional sites of E. coli elongation factor Tu

Recently, we have made significant progress in solving the structure of a nicked form of elongation factor (EF)-Tu complexed with GDP. The structure has been refined to an R factor of 19.2% at 2.6 A resolution, so that most of the structure is clearly visible in the electron density map. Here we describe what is known about functional sites of EF-Tu in terms of the structure, which still lacks amino acids 40-60.

Structural information for explaining the molecular mechanism of protein biosynthesis

Protein biosynthesis is controlled by a number of proteins external to the ribosome. Of these, extensive structural investigations have been performed on elongation factor‐Tu and elongation factor‐G. This now gives a rather complete structural picture of the functional cycle of elongation factor‐Tu and especially of the elongation phase of protein biosynthesis. The discovery that three domains of elongation factor‐G are structurally mimicking the amino‐acylated tRNA in the ternary complex of elongation factor‐Tu has been the basis of much discussion of the functional similarities and functional differences of elongation factor‐Tu and elongation factor‐G in their interactions with the ribosome. Elongation factor‐G:GDP is now thought to leave the ribosome in a state ready for checking the codon‐anticodon interaction of the aminoacyl‐tRNA contained in the ternary complex of elongation factor‐Tu. Elongation factor‐G does this by mimicking the shape of the ternary complex. Other translation factors such as the initiation factor‐2 and the release factor 1 or 2 are also thought to mimic tRNA. These observations raise questions concerning the possible evolution of G‐proteins involved in protein biosynthesis.