Sordarin Derivatives Induce a Novel Conformation of the Yeast Ribosome Translocation Factor eEF2

Rikke Søe,Ralph T Mosley,Michael Justice,Jennifer Nielsen-Kahn,Mythili Shastry,A Rod Merrill,Gregers R Andersen

doi:10.1074/jbc.m607830200

Rikke Søe, Ralph T Mosley + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m607830200

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Abstract

The sordarins are fungal specific inhibitors of the translation factor eEF2, which catalyzes the translocation of tRNA and mRNA after peptide bond formation. We have determined the crystal structures of eEF2 in complex with two novel sordarin derivatives. In both structures, the three domains of eEF2 that form the ligand-binding pocket are oriented in a different manner relative to the rest of eEF2 compared with our previous structure of eEF2 in complex with the parent natural product sordarin. Yeast eEF2 is also shown to bind adenylic nucleotides, which can be displaced by sordarin, suggesting that ADP or ATP also bind to the three C-terminal domains of eEF2. Fusidic acid is a universal inhibitor of translation that targets EF-G or eEF2 and is widely used as an antibiotic against Gram-positive bacteria. Based on mutations conferring resistance to fusidic acid, cryo-EM reconstructions, and x-ray structures of eEF2, EF-G, and an EF-G homolog, we suggest that the conformation of EF-G stalled on the 70 S ribosome by fusidic acid is similar to that of eEF2 trapped on the 80 S ribosome by sordarin.

Highlights

An additional elongation factor, eEF3, is required to facilitate release of the deacylated tRNA from the E-site [2]
This has been confirmed by cryo-EM3 reconstructions of eEF2 in a sordarin-stabilized complex with the 80 S ribosome [8, 9], which is rather similar to the 70 S-EF-G complex stabilized by fusidic acid [10]
The main features of this are that GTP hydrolysis precedes a conformational change of the 70 S-EF-G complex required for translocation

Summary

Data collection Complex Radiation source Space group and unit cell

Resolution (Å) Rsym (%)a Completeness (%) Average I/␴(I) Redundancy eEF2-moriniafungin MAX-LAB 711, l ϭ 1.089 Å P21, a ϭ 59.97 Å, b ϭ 159.12 Å, c ϭ 112.14 Å,. Modified sordarins, including synthetic analogs such as GM 237354 [22] and natural product analogs [24] have enhanced potency and antifungal spectrum of activity. We describe the crystal structures of eEF2 in complex with two modified sordarins, both of which induce a conformation within eEF2 that is quite distinct from that described earlier for eEF2 in the presence of sordarin [25]. Mammalian eEF2 has previously been shown to bind adenylic nucleotides in a site distinct from the well characterized binding site for GDP/GTP [26]. We show that this is a universal feature of eEF2 and that sordarin can displace both ADP and ATP from eEF2. We suggest that the conformation of EF-G on the prokaryotic ribosome targeted by fusidic acid is similar to that of the eEF2-sordarin complex on the eukaryotic ribosome

EXPERIMENTAL PROCEDURES

Findings

DISCUSSION