Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry

Abstract

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39°C, and a minor transition at 58°C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at ~75 kDa and the other at ~55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N- terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C- terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58°C transition, and a loose structure of the C-terminus, giving rise to the 39°C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N- terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA- synthase homologous domain (Glu820-Gly865). Both the basal and ribosome- stimulated ATPase activities were inactivated by trypsin, but the ribosome- stimulated activity was inactivated faster.