Abstract
Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH-S-transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems.
Highlights
Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs
EPRS is the largest human tRNA synthetase, containing an N-terminal ERS domain joined to a C-terminal prolyltRNA synthetase (PRS) domain by an internal linker containing a triple repeat of noncatalytic WHEP domains (Fig. 1A)
Because of the presence of a small amount of aggregates in the ERS2.5W sample analyzed by small-angle neutron scattering (SANS) (Fig. 6A, less than 5%), we evaluated the stability of the P(r) function in the presence and absence of the aggregates
Summary
Site-selective cleavage of EPRS uncouples the glutamyl- and prolyl-tRNA synthetases. EPRS is the largest human tRNA synthetase, containing an N-terminal ERS domain joined to a C-terminal PRS domain by an internal linker containing a triple repeat of noncatalytic WHEP domains (Fig. 1A). In C. elegans, six WHEP repeats are fused to the C terminus of ERS These results suggest that caspase activity may represent an evolutionarily conserved mechanism for inducible separation of ERS and PRS and the generation of novel WHEP domain-containing EPRS proteoforms in mammalian cells. Using purified FLAG-tagged EPRS (Fig. 4C, left panel), recombinant caspase-3 induced robust generation of ERS2.5W, as well as minor fragments possibly produced by cleavage at less preferred sites within ERS (Fig. 4C, right panel). ERS2.5W expression was undetectable in squamous cell carcinoma and advanced-stage colonic adenocarcinoma These results indicate that EPRS cleavage occurs in multiple healthy human tissues in vivo.
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