Ribosomal P0 Protein Domain Involved in Selectivity of Antifungal Sordarin Derivatives

Abstract

The ribosomal stalk protein P0 is involved in the susceptibility to the antifungal sordarin derivatives, as reported for a number of Saccharomyces cerevisiae resistant mutants. Mammals and some lower eukaryotes are naturally resistant to these compounds. It is shown here that expression in S. cerevisiae of the ribosomal protein P0 from Homo sapiens and from other sordarin-resistant organisms results in a decrease in the sensitivity of the cells to an agent of this class. To further characterize the P0 region responsible for inducing sordarin resistance, a series of protein chimeras containing complementary regions of the human and yeast P0 proteins were constructed and expressed in yeast. The chimeras complement the absence of the native yeast P0 except in chimeras containing the human P0 carboxyl-terminal domain. Resistance to sordarins was found to be associated with the presence of an HsP0 amino acid sequence comprising P118 to F138, which unexpectedly led to higher resistance than the presence of the complete human P0. A comparison of the corresponding region in P0 from yeast and sordarin-insensitive organisms, followed by site-directed mutagenesis, indicates that residues in positions 119, 124, and 126 have an important role in determining resistance to sordarins. Moreover, since sordarins block the eukaryotic elongation factor 2 (EF2) function, the P0 region affecting sordarin susceptibility must correspond to EF2-interacting domains of the ribosomal stalk protein, which affects the drug-binding site in the elongation factor.

Structural Basis for Translation Factor Recruitment to the Eukaryotic/Archaeal Ribosomes

“…This is supported by the observation in the cryoelectron microscopy structure of 80S⅐eEF2 complex that eEF2 interacts with this stalk base area (46). This is also consistent with the fact that the insertion domain carries the mutations in Saccharomyces cerevisiae mutants resistant to the antifungal compound sordarin, which acts on eEF2 (47,48).…”

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

“…Additional residues located at other sites in eEF2 have also been implicated in sordarin resistance (5,21). Regarding RPP0, spontaneous resistant mutants have been isolated in the region from amino acid 137 to 144 (13,22), and site-directed mutagenesis results also have implicated residues 119, 124, and 126 of rpP0 in sordarin resistance (39,40). In any case, as sordarin directly binds to eEF2, this factor seems to be the primary target of the drug.…”

Protein–protein interaction map of the Trypanosoma cruzi ribosomal P protein complex

“…The activity of several clinically powerful drugs against microbes is caused by their selective inhibition of protein synthesis (Fostel and Lartey, 2000), by analogy, compounds that may inhibit the parasite protein synthesis are likely to be useful anti-parasitic agents. In this regard, it is noteworthy that sordarin, an anti-fungal drug, seems to act stabilizing the binding of the elongation factor 2 (EF2) to the ribosome, an effect that seems to be enhanced by the presence of the fungal ribosomal P0 protein (Justice et al, 1999;Santos et al, 2004). Perhaps the very specific nature of the ribosomal P protein system of T. cruzi and the proteins that interact with them, L12 and EF2 (Uchiumi and Kominami, 1997;Bargis-Surgey et al, 1999;Lalioti et al, 2002) may enable the development of selective anti-parasite protein synthesis inhibitors.…”