Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains

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 and 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 and 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--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.

Disease-associated mutations in a bifunctional aminoacyl-tRNA synthetase gene elicit the integrated stress response

“…PRS displayed robust in vitro amino acid activation and aminoacylation activity (34). In contrast, recombinant ERS proteins with part of the linker region were purified and showed moderate aminoacylation activity (35). In our hands, bacterial expression of an ERS-only construct containing residues 1-687 of human EPRS resulted in the protein appearing in inclusion bodies.…”

“…To remedy this expression problem, we designed two maltosebinding protein (MBP)-tagged extended ERS constructs: MBP-ERSRC (residues 1-749), which included amino acid sequences at the C-terminus of the ERS domain that are in a random coil (RC) region prior to the WHEP domains, and MBP-ERS2.5W (residues 1-929) with the RC and 2.5 WHEP domains (Figure 1A). The ERS2.5W protein was previously found to be present in cells as a caspase-3 cleavage product (35). The final purified proteins were estimated to be ~90% pure (Figure S2B).…”

“…While no successful purification of full-length human EPRS protein has been reported, recombinant ERS fragments of different lengths have been purified and displayed only modest levels of activity (16,35). Whereas the MBP-ERS protein lacking any linker residues appeared in inclusion bodies (data not shown), the MBP-ERSRC and MBP-ERS2.5W recombinant proteins were highly soluble, suggesting that the linker region facilitates proper folding of the ERS catalytic core.…”

Hypomyelinating Leukodystrophy 15 (HLD15)-Associated Mutation of EPRS1 Leads to Its Polymeric Aggregation in Rab7-Positive Vesicle Structures, Inhibiting Oligodendroglial Cell Morphological Differentiation

“…Human EPRS1 contains the D 926 XXD 929 motif, which is cleaved by a caspase enzyme [25]. This motif is conserved among humans and other mammalian species such as mice and rats.…”

“…After the protein has been cleaved, the N-terminal half has only the catalytic activity of glutamyl-tRNA synthetase (EARS), while the C-terminal half has only the catalytic activity of prolyl-tRNA synthetase (PARS). These caspase-cleaved EPRS1 halfproteins can also be incorporated into the functional tRNA multisynthetase complex (MSC), which is composed of aminoacyl-tRNA synthetases along with other adaptor proteins [25], suggesting that both the EARS unit and the PARS unit of EPRS1 are structurally and functionally required for MSC. Indeed, wild-type EPRS1 supports both EARS and PARS activities in cells.…”