Uncovering of a Short Internal Peptide Activates a tRNA Synthetase Procytokine

Abstract

Background: Many tRNA synthetases have acquired expanded functions unrelated to their canonical aminoacylation function. Results: Structural analysis in solution showed that a subtle conformational change causes pinpointed exposure of an evolutionarily acquired ELR tripeptide of human tyrosyl-tRNA synthetase, which activates its cytokine function. Conclusion:The exposure of ELR of TyrRS confers cytokine activity. Significance: The TyrRS design facilitates functional expansion while retaining its essential role in translation.In higher organisms, aminoacyl-tRNA synthetases developed receptor-mediated ex-translational functions that are activated by various natural mechanisms. Hydrogen-deuterium exchange combined with mass spectrometry and small-angle x-ray scattering showed that activation of the cytokine function of the 528-amino acid human tyrosyl-tRNA synthetase was associated with pinpointed uncovering of a miniature internal ELR tripeptide that triggers receptor signaling. The results reveal the structural simplicity of how the ex-translational function is implemented.Aminoacyl-tRNA synthetases are ancient enzymes that catalyze the first step of protein synthesis by attaching amino acids onto their cognate tRNAs (1). In addition, in higher organisms, aminoacyl-tRNA synthetases gained a variety of expanded functions in cell signaling pathways ranging from vascular to inflammatory pathways, among others (2-6). These expansions, often implemented with secreted forms, developed in evolution through the incorporation of novel domains and sequence motifs that bear no impact on the protein synthesis function. That these additions were added at precise time points in evolution and are tightly regulated suggests that they played a role in the development of higher organisms (7-9).Human tyrosyl-tRNA synthetase (TyrRS) 2 is a 528-amino acid procytokine composed of two domains connected by a long linker that spans ϳ20 amino acids (Fig. 1A). The 364-amino acid N-terminal part (mini-TyrRS) is the catalytic aminoacylation domain and is active without the C-terminal domain (CTD) (10,11). At the evolutionary stage of insects, an ELR tripeptide was incorporated into the mini-TyrRS domain and has been retained in higher organisms. The ELR motif is the recognition element within CXC chemokines such as interleukin-8, which mediates the innate immune response as well as angiogenesis (12,13). Simultaneously, the CTD, which is a structural and functional homolog of human endothelial monocyte-activating polypeptide II (EMAP II), was appended to TyrRS. Previous work established that mini-TyrRS and the CTD have distinct cytokine activities associated with hematological and inflammatory pathways, whereas full-length TyrRS is inactive as a cytokine (14,15). Thus, the novel activities are observed only when the two domains of secreted TyrRS are separated by natural proteolysis at the interdomain linker.The crystal structures of mini-TyrRS and the CTD have separately been determined (16,17). In the mini-TyrRS domain, the ELR motif is mask...

Internally Deleted Human tRNA Synthetase Suggests Evolutionary Pressure for Repurposing

“…These activities include major roles in regulating angiogenesis (Xu et al, 2012; Yao et al, 2012), inflammatory responses (Arif et al, 2009; Fu et al, 2012; Lee et al, 2012), mTOR signaling (Bonfils et al, 2012; Han et al, 2012), and tumor growth (Dorrell et al, 2007; Park et al, 2012). In at least some instances, a fragment produced by natural proteolysis is the active factor.…”

A human tRNA synthetase is a potent PARP1-activating effector target for resveratrol

“…2c , left). By breaking the Y341 OH--H-bond to a backbone carbonyl oxygen, a Y341A mutation releases tight tethering of the C-domain to the catalytic domain 15 and its reorientation 16 ( Fig. 2c , right).…”

Applications of Hydrogen/Deuterium Exchange MS from 2012 to 2014

“…Analysis of the Y341A mutant of human tyrosyl-tRNA synthetase, a functional mutant that would not crystallize, with HDX MS and SAXS revealed conformational changes essential for activation via uncovering of an internal tripeptide sequence. 50 In the seryl-tRNA synthetase, HDX MS was used 51 to understand why a mutation (F383V) located ∼100 amino acids upstream of the nuclear localization signal (NLS) somehow abolished nuclear localization. The NLS was not resolved in a crystal of the protein, but HDX MS showed that the NLS was protected from exchange in the F383 V mutant versus the wild-type.…”