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

Jørgensen, René; Ortiz, Pedro A.; Carr-Schmid, Anne; Nissen, Poul; Kinzy, Terri Goss; Andersen, G.R.

doi:10.1038/nsb923

Cited by 159 publications

(214 citation statements)

References 40 publications

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“…In contrast to EF-Tu, EF-G contains a GЈ subdomain, which is invariably inserted between ␣-helices D G and E G (5). The GЈ subdomain is also present in the same location in EF-2, the eukaryotic cytoplasmic homolog of EF-G (6). However, the GЈ subdomains of EF-G and EF-2 are unrelated in their amino acid sequences and tertiary structures.…”

Section: Together These Results Provide Evidence For Functionally Immentioning

confidence: 91%

“…Experiments in which L7/L12 was replaced with P proteins on the E. coli ribosome demonstrated functionality of the eukaryotic factors on these hybrid ribosomes (38). Considered in light of our present results, we speculate that the specificity determinants of EF-G and EF-2 may involve the GЈ subdomains of these factors, which have unrelated sequences and tertiary structures (6). The specificity determinants of EF-Tu and EF-1␣ remain unknown, but it is interesting to note that their G domains differ by the addition or deletion of several ␣-helices (39), and their shared D G helices have different sequences.…”

Section: Discussionmentioning

confidence: 90%

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Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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Protein L7/L12 of the bacterial ribosome plays an important role in activating the GTP hydrolytic activity of elongation factor G (EF-G), which promotes ribosomal translocation during protein synthesis. Previously, we cross-linked L7/L12 from two residues (209 and 231) flanking ␣-helix A G in the G subdomain of Escherichia coli EF-G. Here we report kinetic studies on the functional effects of mutating three neighboring glutamic acid residues (224, 228, and 231) to lysine, either singly or in combination. Two single mutations (E224K and E228K), both within helix A G , caused large defects in GTP hydrolysis and smaller defects in ribosomal translocation. Removal of L7/L12 from the ribosome strongly reduced the activities of wild type EF-G but had no effect on the activities of the E224K and E228K mutants. Together, these results provide evidence for functionally important interactions between helix A G of EF-G and L7/L12 of the ribosome.

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Section: Together These Results Provide Evidence For Functionally Immentioning

confidence: 91%

Section: Discussionmentioning

confidence: 90%

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Nechifor

Murataliev

Wilson

2007

Journal of Biological Chemistry

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“…In addition, the structures of prokaryotic (formerly termed EF-G) 23 and eukaryotic EF2 24 have also been elucidated, revealing an overall shape similarity ("macromolecular mimicry") 25 between EF2‚GDP and EF1A‚GTP‚tRNA.…”

Section: Introductionmentioning

confidence: 99%

Structural Basis for the Binding of Didemnins to Human Elongation Factor eEF1A and Rationale for the Potent Antitumor Activity of These Marine Natural Products

et al. 2004

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Didemnins and tamandarins are closely related marine natural products with potent inhibitory effects on protein synthesis and cell viability. On the basis of available biochemical and structural evidence and results from molecular dynamics simulations, a model is proposed that accounts for the strong and selective binding of these compounds to human elongation factor eEF1A in the presence of GTP. We suggest that the p-methoxyphenyl ring of these cyclic depsipeptides is inserted into the same pocket in eEF1A that normally lodges either the 3′ terminal adenine of aminoacylated tRNA, as inferred from two prokaryotic EF-Tu‚GTP‚tRNA complexes, or the aromatic side chain of Phe/Tyr-163 from the nucleotide exchange factor eEF1BR, as observed in several X-ray crystal structures of a yeast eEF1A:eEF1BR complex. This pocket, which has a strong hydrophobic character, is formed by two protruding loops on the surface of eEF1A domain 2. Further stabilization of the bound depsipeptide is brought about by additional crucial interactions involving eEF1A domain 1 in such a way that the molecule fits snugly at the interface between these two domains. In the GDP-bound form of eEF1A, this binding site exists only as two separate halves, which accounts for the much greater affinity of didemnins for the GTP-bound form of this elongation factor. This binding mode is entirely different from those seen in the complexes of the homologous prokaryotic EF-Tu with kirromycin-type antibiotics or the cyclic thiazolyl peptide antibiotic GE2270A. Interestingly, the set of interactions used by didemnins to bind to eEF1A is also distinct from that used by eEF1BR or eEF1B , thus establishing a competition for binding to a common site that goes beyond simple molecular mimicry. The model presented here is consistent with both available biochemical evidence and known structure-activity relationships for these two classes of natural compounds and synthetic analogues and provides fertile ground for future research.

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“…The general problem appears to be the toxicity of high selenium concentrations to eukaryotic cells, which is somewhat surprising, because SeMet has been shown previously to be functional when incorporated into (S)-adenosylmethionine (7). The use of an industrial strain of yeast allowed the preparation of a SeMet derivative of translation elongation factor 2 (8). However, although this approach works for a very highly expressed endogenous protein, because of the difficulty in applying molecular genetic approaches in industrial strains it is not applicable to conditions where the chromosomal gene encoding the protein must be deleted or the protein of interest must be expressed from a plasmid.…”

mentioning

confidence: 99%

Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

Bockhorn¹,

Balar²,

He³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Selenomethionine (SeMet) is a potentially toxic amino acid, and yet it is a valuable tool in the preparation of labeled proteins for multiwavelength anomalous dispersion or single-wavelength anomalous dispersion phasing in X-ray crystallography. The mechanism by which high levels of SeMet exhibits its toxic effects in eukaryotic cells is not fully understood. Attempts to use Saccharomyces cerevisiae for the preparation of fully substituted SeMet proteins for X-ray crystallography have been limited. A screen of the viable S. cerevisiae haploid null allele strain collection for resistance to SeMet was performed. Deletion of the CYS3 gene encoding cystathionine gamma-lyase resulted in the highest resistance to SeMet. In addition, deletion of SSN2 resulted in both increased resistance to SeMet as well as reduced levels of Cys3p. A methionine auxotrophic strain lacking CYS3 was able to grow in media with SeMet as the only source of Met, achieving essentially 100% occupancy in total proteins. The CYS3 deletion strain provides advantages for an easy and cost-effective method to prepare SeMet-substituted protein in yeast and perhaps other eukaryotic systems.CYS3 ͉ SSN2

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

Cited by 159 publications

References 40 publications

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Functional Interactions between the G′ Subdomain of Bacterial Translation Factor EF-G and Ribosomal Protein L7/L12

Structural Basis for the Binding of Didemnins to Human Elongation Factor eEF1A and Rationale for the Potent Antitumor Activity of These Marine Natural Products

Genome-wide screen of Saccharomyces cerevisiae null allele strains identifies genes involved in selenomethionine resistance

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