Orthogonal use of a human tRNA synthetase active site to achieve multifunctionality

Abstract

Protein multi-functionality is an emerging explanation for the complexity of higher organisms. In this regard, while aminoacyl tRNA synthetases catalyze amino acid activation for protein synthesis, some also act in pathways for inflammation, angiogenesis, and apoptosis. How multiple functions evolved and their relationship to the active site is not clear. Here structural modeling analysis, mutagenesis, and cell-based functional studies show that the potent angiostatic, natural fragment of human TrpRS associates via Trp side chains that protrude from the cognate cellular receptor VE-cadherin. Modeling indicates that (I prefer the way it was because the conclusion was reached not only by modeling, but more so by experimental studies.)VE-cadherin Trp side chains fit into the Trp-specific active site of the synthetase. Thus, specific side chains of the receptor mimic (?) amino acid substrates and expand the functionality of the active site of the synthetase. We propose that orthogonal use of the same active site may be a general way to develop multifunctionality of human tRNA synthetases and other proteins.Along with alternative splicing, post-translational modifications, and regulatory RNAs, protein multi-functionality is an emerging explanation for the complexity of higher organisms. Multi-functionality reduces the need for more protein-coding genes. In addition, expanded functions provide a way to integrate diverse biological systems, where one pathway is connected to another. Prominent among the various examples of multifunctional proteins are members of the aminoacyl tRNA synthetase (AARS) family 1, 2. While AARSs are well known for activating amino acids for protein synthesis 3, they also have a broad spectrum of expanded activities in pathways including splicing 4 ,5 , translational and transcriptional control 6-8 , pro-and anti-angiogenesis 9-14 , inflammation 15 , signaling of immune responses 16 , apoptosis 17 , and viral assembly 18 . It is these activities that may explain why there are many disease-associated tRNA synthetase mutations, especially in instances where the change has no effect on aminoacylation activity [19][20][21][22][23] . In most cases, Correspondence: xlyang@scripps.edu, Phone: (858) however, how and why tRNA synthetases were expropriated for other purposes is still not understood.Upon stimulation with the anti-proliferative cytokine interferon-γ, the 471 amino acid human tryptophanyl-tRNA synthetase (TrpRS) is up-regulated and secreted from various cell types including endothelial cells 24, 25. Natural alternative forms of human TrpRS have potent anti-angiogenic activity 9 -11 ,14 . This activity is blocked until the N-terminal domain (N-domain) is removed, either by alternative splicing to give mini-TrpRS, or by proteolysis, to give the closely similar T2-TrpRS (comprised of residues 94-471 of TrpRS). T2-TrpRS binds to the endothelial cell adherens junction molecule-vascular endothelial-cadherin (VE-cadherin) 26 -and inhibits activation of genes associated with angiogenesis ...

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

“…6C). As expected from previous studies, 8 T2-TrpRS pulled down substantially larger amounts of VE-cadherin than did the FL-TrpRS. Interestingly, the H130R mutation enhanced the VE-cadherin interaction, especially in the context of T2-TrpRS.…”

“…Considering the importance of the Trp and AMP binding pockets for the T2-TrpRS and VE-cadherin interaction, 8 we evaluated how the H130R mutation would affect the interaction using molecular docking and dynamic simulations (MDS). We used the crystal structure of the EC1-2 domain of chicken VE-cadherin 27 (PDB ID 3PPE) that share 58% sequence identity with human VE-cadherin to docked onto WT and H130R T2-TrpRS structures, respectively.…”

“…7,8,9 Our previous study has demonstrated that T2-TpRS binds the two tryptophan residues (Trp2 and Trp4) through its tryptophan and AMP binding pockets in the active site, respectively, and thereby inhibits the VE-cadherin mediated endothelial cell-cell junctions and disrupts the formation of new blood vessels. 8 Notably, human TrpRS manifests its anti-angiogenic activity only when the WHEP domain is removed. Full length (FL) TrpRS cannot inhibit angiogenesis, 5,6,10 due to the steric hindrance of the WHEP domain that blocks the access of Trp2 and Trp4 of VE-cadherin to the active site pocket.…”

“…Full length (FL) TrpRS cannot inhibit angiogenesis, 5,6,10 due to the steric hindrance of the WHEP domain that blocks the access of Trp2 and Trp4 of VE-cadherin to the active site pocket. 8 …”

Upregulation of tryptophanyl-tRNA synthethase adapts human cancer cells to nutritional stress caused by tryptophan degradation

“…A truncated version of WARS is secreted in response to IFNγ and elicits angiostatic effects 23 through inhibition of endothelial cell-cell junctions 24 . Upon infection, monocytes secrete full-length WARS, which induces phagocytosis and chemokine production in macrophages 21 .…”