Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2 The GenBank accession numbers for the sequences reported in this manuscript are AF107286–AF107291, AF292693 and AF248644.

Abstract

The sordarin class of natural products selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Mutations in Saccharomyces cerevisiae eEF2 or the ribosomal stalk protein rpP0 can confer resistance to sordarin, although eEF2 is the major determinant of sordarin specificity. It has been shown previously that sordarin specifically binds S. cerevisiae eEF2 while there is no detectable binding to eEF2 from plants or mammals, despite the high level of amino acid sequence conservation among these proteins. In both whole-cell assays and in vitro translation assays, the efficacy of sordarin varies among different species of pathogenic fungi. To investigate the basis of sordarin's fungal selectivity, eEF2 has been cloned and characterized from several sordarin-sensitive and -insensitive fungal species. Results from in vivo expression of Candida species eEF2s in S. cerevisiae and in vitro translation and growth inhibition assays using hybrid S. cerevisiae eEF2 proteins demonstrate that three amino acid residues within eEF2 account for the selectivity of this class of compounds. It is also shown that the corresponding residues at these positions in human eEF2 are sufficient to confer sordarin insensitivity to S. cerevisiae identical to that observed with mammalian eEF2.

Identification and Characterization of a Novel Evolutionarily Conserved Lysine-specific Methyltransferase Targeting Eukaryotic Translation Elongation Factor 2 (eEF2)

“…This indicates that the methylation state of Lys-509 affects the interaction of S. cerevisiae eEF2 with sordarin, and, accordingly, sensitivity toward this drug has been found to be determined by the sequence of amino acids in the "sordarin specificity region" (residues 517-524 in S. cerevisiae eEF2) at the entrance to the sordarin binding cleft (47), which is in close proximity to the Lys-509 in the three-dimensional structure of eEF2 (39).…”

A Chemical Genomic Screen in Saccharomyces cerevisiae Reveals a Role for Diphthamidation of Translation Elongation Factor 2 in Inhibition of Protein Synthesis by Sordarin

“…Two proteins have been described as targets of sordarin and its derivatives, both involved in protein synthesis: translation elongation factor 2 (eEF2) (5,7,21) and the large ribosomal subunit protein rpP0 (13,22). In the former target, characterization in yeast (Saccharomyces cerevisiae) of spontaneous resistant mutants along with site-directed mutagenesis has been used to identify residues 521, 523, and 524 as the most important amino acids in eEF2 involved in the binding of sordarin (41). Additional residues located at other sites in eEF2 have also been implicated in sordarin resistance (5,21).…”

Applications of yeast in drug discovery

“…The ability to inhibit protein synthesis in these organisms correlated well with inhibition of fungal growth and suggests that resistance to sordarins in specific organisms is due to intrinsic differences in the molecular target. Shastry et al [100] isolated and expressed EF2s from various Candida species, including those sensitive and intrinsically resistant to sordarins, in S. cerevisiae. Results from in vitro translation and growth inhibition assays showed that residues in an 8-amino acid region (517-524), in particular residues 521, 523 and 524, within EF2 are responsible for the selectivity of this class of compounds [100].…”

“…Shastry et al [100] isolated and expressed EF2s from various Candida species, including those sensitive and intrinsically resistant to sordarins, in S. cerevisiae. Results from in vitro translation and growth inhibition assays showed that residues in an 8-amino acid region (517-524), in particular residues 521, 523 and 524, within EF2 are responsible for the selectivity of this class of compounds [100]. The difference between S. cerevisiae and the human in the corresponding residues likewise accounts for the insensitivity of the human protein to sordarins, a conclusion that could not have been reached without the precise molecular details afforded by S. cerevisiae.…”