Crystal structure of a mutant elongation factor G trapped with a GTP analogue

Hansson, Sebastian; Singh, Ranvir; Gudkov, A.T.; Liljas, Anders; Logan, D.T.

doi:10.1016/j.febslet.2005.07.016

Cited by 69 publications

(89 citation statements)

References 41 publications

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“…S2A). This finding is not entirely surprising given that the structure of EF-G in nucleotide-free form (PDB ID code 1ELO) is almost identical to that of GTP-(2BV3) and GDP-(2BM0) bound forms (27,29,30). Nevertheless, the structure of isolated BipA bound to GDPCP (a GTP analog) allowed us to delineate the nucleotide-binding site (SI Results).…”

Section: Resultsmentioning

confidence: 93%

Structure of BipA in GTP form bound to the ratcheted ribosome

Kumar

Chen

Ero

et al. 2015

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

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BPI-inducible protein A (BipA) is a member of the family of ribosome-dependent translational GTPase (trGTPase) factors along with elongation factors G and 4 (EF-G and EF4). Despite being highly conserved in bacteria and playing a critical role in coordinating cellular responses to environmental changes, its structures (isolated and ribosome bound) remain elusive. Here, we present the crystal structures of apo form and GTP analog, GDP, and guanosine-3′,5′-bisdiphosphate (ppGpp)-bound BipA. In addition to having a distinctive domain arrangement, the C-terminal domain of BipA has a unique fold. Furthermore, we report the cryo-electron microscopy structure of BipA bound to the ribosome in its active GTP form and elucidate the unique structural attributes of BipA interactions with the ribosome and A-site tRNA in the light of its possible function in regulating translation.

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Section: Resultsmentioning

confidence: 93%

Structure of BipA in GTP form bound to the ratcheted ribosome

Kumar

Chen

Ero

et al. 2015

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

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“…The conformation of EF-G bound to FA on the ribosome has been characterized by cryo-EM of ribosome-EF-G complexes stalled by FA (Agrawal et al 1998;Stark et al 2000). Compared to the conformation of unbound EF-G, determined crystallographically (AEvarsson et al 1994;Czworkowski et al 1994;Al-Karadaghi et al 1996;Hansson et al 2005), in the ribosome-bound conformation domain IV of EF-G is tilted relative to the body of the molecule formed of domains I and II. Based on cryo-EM, it has been suggested that the rearrangement of EF-G that is induced by GTP hydrolysis consists of a movement of domain IV.…”

Section: Ribosome Disassembly As An In Vivo Target Of Fusidic Acidmentioning

confidence: 99%

Distinct functions of elongation factor G in ribosome recycling and translocation

Savelsbergh

Rodnina

Wintermeyer

2009

RNA

117

135

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Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.

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“…However, the question of whether different nucleotides or nucleotide analogs exhibit different properties for assembly of the SRP GTPase complex has not been fully explored. Interaction specific to the analog GMPPNP in a different GTPase was suggested by a structure of an EF-G complex in which a peptide flip within the P-loop directs a carbonyl oxygen towards the β-γ amido group of the analog (Hansson et al, 2005). A peptide flip of the corresponding residue of the P-loop in Ffh is observed as a minor population in structures of the Ffh NG domain determined ultra-high resolution (Ramirez and Freymann, 2006).…”

mentioning

confidence: 99%

Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY

Gawronski-Salerno

Freymann

2007

Journal of Structural Biology

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Ffh and FtsY are GTPase components of the signal recognition particle co-translational targeting complex that assemble during the SRP cycle to form a GTP-dependent and pseudo two-fold symmetric heterodimer. Previously the SRP GTPase heterodimer has been stabilized and purified for crystallographic studies using both the non-hydrolysable GTP analog GMPPCP and the pseudotransition state analog GDP:AlF 4 , revealing in both cases a buried nucleotide pair that bridges and forms a key element of the heterodimer interface. A complex of Ffh and FtsY from T. aquaticus formed in the presence of the analog GMPPNP could not be obtained, however. The origin of this failure was previously unclear, and it was thought to have arisen from either instability of the analog, or, alternatively, from differences in its interactions within the tightly conscribed composite active site chamber of the complex. Using insights gained from the previous structure determinations, we have now determined the structure of the SRP GTPase targeting heterodimer stabilized by the nonhydrolysable GTP analog GMPPNP. The structure demonstrates how the different GTP analogs are accommodated within the active site chamber despite slight differences in the geometry of the phosphate chain. It also reveals a K + coordination site at the highly conserved DARGG loop at the N/G interdomain interface.The Signal Recognition Particle (SRP) mediates signal peptide recognition and co-translational targeting of secreted and membrane proteins to the membrane translocon (Walter and Johnson, 1994;Keenan et al., 2001). SRP is a phylogenetically conserved ribonucleoprotein that comprises, in prokaryotes, Ffh, the SRP GTPase, and the 4.5S RNA (Luirink and Dobberstein, 1994). The primary structure of Ffh includes three domains, the N and G domains, and the M domain (Bernstein et al., 1989;Römisch et al., 1989). The M domain provides sites for signal sequence recognition and for interaction with the RNA (Zopf et al., 1990;Luirink et al., 1992;Lütcke et al., 1992). The N and G domains of the SRP GTPase, together the 'NG' domain, form a structural and functional unit (Freymann et al., 1997). The membrane associated receptor for SRP is also phylogenetically conserved, and its primary structure includes an NG GTPase as well (Montoya et al., 1997). The two SRP NG GTPases interact directly with each other forming a GTP-dependent heterodimeric targeting complex that plays a central role in co-translational protein targeting to the membrane (Powers and Walter, 1995;Powers and Walter, 1997;Rapiejko and Gilmore, 1997;Song et al., 2000;Mandon et al., 2003). * corresponding author: phone: 312/503-1877, fax: 312/503-5349, email: freymann@northwestern.edu. Author Contributions J.G.S. purified the GMPPNP-stabilized complex and setup the crystallization screen; D.M.F. carried out the crystallographic work and wrote the manuscript.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providi...

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Crystal structure of a mutant elongation factor G trapped with a GTP analogue

Cited by 69 publications

References 41 publications

Structure of BipA in GTP form bound to the ratcheted ribosome

Structure of BipA in GTP form bound to the ratcheted ribosome

Distinct functions of elongation factor G in ribosome recycling and translocation

Structure of the GMPPNP-stabilized NG domain complex of the SRP GTPases Ffh and FtsY

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