Crystal Structure of Intact Elongation Factor EF-Tu from Escherichia coli in GDP Conformation at 2.05Å Resolution

Song, Haiwei; Parsons, Mark R.; Rowsell, Siân; Leonard, Gordon A.; Phillips, Simon E. V.

doi:10.1006/jmbi.1998.2387

Cited by 134 publications

(166 citation statements)

References 33 publications

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“…Specific molecular interactions and processes have been assigned to many parts of the three domains of the protein (Krab and Parmeggiani, 1998). The function of the N terminus, however, remains largely unexplained, and x-ray crystallography did not reveal a clear structure for the seven amino acids at the N terminus of the protein (Song et al, 1999). Nevertheless, this part of the protein is equally highly conserved, notably for the basic residues and the Phe found to be relevant also for elicitor activity, suggesting an essential function also for this part of the EF-Tu (Laurberg et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

et al. 2004

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Innate immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs). Here, we show that elongation factor Tu (EF-Tu), the most abundant bacterial protein, acts as a PAMP in Arabidopsis thaliana and other Brassicaceae. EF-Tu is highly conserved in all bacteria and is known to be N-acetylated in Escherichia coli. Arabidopsis plants specifically recognize the N terminus of the protein, and an N-acetylated peptide comprising the first 18 amino acids, termed elf18, is fully active as inducer of defense responses. The shorter peptide, elf12, comprising the acetyl group and the first 12 N-terminal amino acids, is inactive as elicitor but acts as a specific antagonist for EF-Tu–related elicitors. In leaves of Arabidopsis plants, elf18 induces an oxidative burst and biosynthesis of ethylene, and it triggers resistance to subsequent infection with pathogenic bacteria

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Section: Discussionmentioning

confidence: 99%

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

et al. 2004

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“…Correct codon-anticodon recognition greatly stabilizes the ternary complex on the ribosome and activates GTP hydrolysis. Following formation of GDP and subsequent P i release (5, 6), EF-Tu undergoes a large conformational change (7,8) causing the release of the bound aa-tRNA followed by accommodation of the aa-tRNA into the 50S ribosomal A site. The intrinsic rate of GDP dissociation from E. coli EF-Tu is extremely slow (0.002 s Ϫ1 (9)), requiring the guanine nucleotide exchange factor EF-Ts (10) to facilitate the rapid conversion of EF-Tu⅐GDP into its active GTP-bound state and to maintain rates of protein synthesis (ϳ12 amino acids/s (11)) observed in vivo.…”

Section: Elongation Factor (Ef)mentioning

confidence: 99%

The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange

Laurentiis

Mercier

Wieden

2016

Journal of Biological Chemistry

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Little is known about the conservation of critical kinetic parameters and the mechanistic strategies of elongation factor (EF) Ts-catalyzed nucleotide exchange in EF-Tu in bacteria and particularly in clinically relevant pathogens. EF-Tu from the clinically relevant pathogen Pseudomonas aeruginosa shares over 84% sequence identity with the corresponding elongation factor from Escherichia coli. Interestingly, the functionally closely linked EF-Ts only shares 55% sequence identity. To identify any differences in the nucleotide binding properties, as well as in the EF-Ts-mediated nucleotide exchange reaction, we performed a comparative rapid kinetics and mutagenesis analysis of the nucleotide exchange mechanism for both the E. coli and P. aeruginosa systems, identifying helix 13 of EF-Ts as a previously unnoticed regulatory element in the nucleotide exchange mechanism with species-specific elements. Our findings support the base side-first entry of the nucleotide into the binding pocket of the EF-Tu⅐EF-Ts binary complex, followed by displacement of helix 13 and rapid binding of the phosphate side of the nucleotide, ultimately leading to the release of EF-Ts. Elongation factor (EF)2 Tu is an essential protein present in all kingdoms of life. It is among the most abundant proteins in any given cell, representing up to 5% of the total cellular protein in Escherichia coli (1). In its active GTP-bound state, EF-Tu delivers aminoacyl-tRNA (aa-tRNA) to actively translating ribosomes in a codon-dependent manner (2). EF-Tu⅐GTP has a high affinity for aa-tRNA (K D Ϸ 1 ϫ 10 Ϫ8 M (3)) and forms a ternary complex that can interact with the ribosome (4). Correct codon-anticodon recognition greatly stabilizes the ternary complex on the ribosome and activates GTP hydrolysis. Following formation of GDP and subsequent P i release (5, 6), EF-Tu undergoes a large conformational change (7,8) causing the release of the bound aa-tRNA followed by accommodation of the aa-tRNA into the 50S ribosomal A site. The intrinsic rate of GDP dissociation from E. coli EF-Tu is extremely slow (0.002 s Ϫ1 (9)), requiring the guanine nucleotide exchange factor EF-Ts (10) to facilitate the rapid conversion of EF-Tu⅐GDP into its active GTP-bound state and to maintain rates of protein synthesis (ϳ12 amino acids/s (11)) observed in vivo. Despite the critical cellular role of EF-Ts, its sequence is highly divergent among different bacterial species. Poor conservation of the primary sequence within bacterial EF-Ts sequences may give rise to differences in the enzymatic properties, raising the question of how and if the enzymatic properties are maintained among different bacterial EF-Tu⅐EF-Ts pairs. This is particularly interesting because the structure of the eukaryotic exchange factor is completely unrelated to its bacterial counterpart, whereas the respective structure for eEF1A remains similar to EF-Tu (12).Based on the experimental approach used in previous studies to dissect the kinetic details of the interaction between EF-Tu, EF-Ts, and the functionall...

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“…In many crystallographic structures the positions of structural water molecules are resolved together with the protein. In the structure of the whole EFTu 66 and EF-1α 67 proteins, a few molecules are visible in the interior of each G-domain. However, these structures were resolved in the presence of substrates which possibly make inaccessible alternative sites.…”

Section: The Exchange Kinetics Of Internal Water Moleculesmentioning

confidence: 99%

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

et al. 2014

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In this work we address the question whether the enhanced stability of thermophilic proteins has a direct connection with internal hydration. Our model systems are two homologues G-domains of different stability: the mesophilic G domain of the Elongation-Factor thermal unstable protein from E. coli and the hyperthermophilic G domain of the EF-α protein from S. solfataricus. Using molecular dynamics simulation at the microsecond time-scale we show that both proteins host water molecules in internal cavities and that these molecules exchange with the external solution in the nanosecond time scale. The hydration free energy of these sites evaluated via extensive calculations is found to be favourable for both systems, with the hyperthermophilic protein offering a slightly more favourable environment to host water molecules. We estimate that under ambient conditions, the free energy gain due to internal hydration is about 1.3 kcal/mol in favor of the hyperthermophilic variant. However, we also find that at the high working temperature of the hyperthermophile, the cavities are rather dehydrated, meaning that at extreme conditions other molecular factors secure the stability of the protein. Interestingly, we detect a clear correlation between the hydration of internal cavities and the protein conformational landscape. The emerging picture is that internal hydration is an effective observable to probe the conformational landscape of proteins. In the specific context of our investigation, the analysis confirms that the hyperthermophilic G-domain is characterized by multiple states and it has a more flexible structure than its mesophilic homologue.

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Crystal Structure of Intact Elongation Factor EF-Tu from Escherichia coli in GDP Conformation at 2.05Å Resolution

Cited by 134 publications

References 33 publications

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants

The C-terminal Helix of Pseudomonas aeruginosa Elongation Factor Ts Tunes EF-Tu Dynamics to Modulate Nucleotide Exchange

Role of Internal Water on Protein Thermal Stability: The Case of Homologous G Domains

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