Crystal structure of a human aminoacyl-tRNA synthetase cytokine

Abstract

The 20 aminoacyl-tRNA synthetases catalyze the first step of protein synthesis and establish the rules of the genetic code through aminoacylation reactions. Biological fragments of two human enzymes, tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, connect protein synthesis to cell-signaling pathways including angiogenesis. Alternative splicing or proteolysis produces these fragments. The proangiogenic N-terminal fragment mini-TyrRS has IL-8-like cytokine activity that, like other CXC cytokines, depends on a Glu-Leu-Arg motif. Point mutations in this motif abolish cytokine activity. The full-length native TyrRS lacks cytokine activity. No structure has been available for any mammalian tRNA synthetase that, in turn, might give insight into why mini-TyrRS and not TyrRS has cytokine activities. Here, the structure of human mini-TyrRS, which contains both the catalytic and the anticodon recognition domain, is reported to a resolution of 1.18 Å. The critical Glu-Leu-Arg motif is located on an internal ␣-helix of the catalytic domain, where the guanidino side chain of R is part of a hydrogen-bonding network tethering the anticodonrecognition domain back to the catalytic site. Whereas the catalytic domains of the human and bacterial enzymes superimpose, the spatial disposition of the anticodon recognition domain relative to the catalytic domain is unique in mini-TyrRS relative to the bacterial orthologs. This unique orientation of the anticodon-recognition domain can explain why the fragment mini-TyrRS, and not fulllength native TyrRS, is active in cytokine-signaling pathways.

Structural basis of nonnatural amino acid recognition by an engineered aminoacyl-tRNA synthetase for genetic code expansion

“…To date, a number of 3D structures of native TyrRS molecules have been determined (13)(14)(15)(16)(17)(18)(19), revealing the substrate recognition mechanisms of the natural enzymes. In the present study, we solved the structures of the 37V195C mutant E. coli TyrRS catalytic domain (19) complexed with 3-iodo-L-tyrosine and with the misrecognized ligand, L-tyrosine, at resolutions of 2.0 Å.…”

Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains

“…Human TrpRS was prepared as described (15) and maintained in a stock solution of 10 mM Hepes (pH 7.5), 20 mM KCl, 0.02% NaN 3 , 2 mM 2-mercaptoethanol. Initial crystallization trials were conducted by using the proprietary high throughput protein crystallization platform developed at Syrrx, Inc. (La Jolla, CA) as described (14). Single crystals were obtained by vapor diffusion of sitting drops (2 l of protein sample and 2 l of reservoir solution) against a reservoir of 2% PEG 8000, 0.1 M Mes͞Mes-Na (pH 6.32) at 4°C.…”

“…The crystal diffracted to 1.6 Å and had the same space group and similar lattice as for miniTyrRS alone (14). The asymmetric unit comprised one monomer of the dimeric complex.…”

“…To locate definitively the amino acid-binding pocket, a cocrystal structure of human miniTyrRS with the amino acid analog tyrosinol was also obtained. Except for the active site residues involved in amino acid binding, miniTyrRS in the complex has essentially the same conformation as in the unligand form (14). Superpositions of TrpRS and miniTyrRS structures from human and the published TyrRS (9,11,12) and TrpRS (10) structures from bacteria together enabled accurate alignment of over 90 sequences in their common core elements.…”

“…In addition, we solved the crystal structure of human TrpRS with bound tryptophanyl-adenylate (Trp-AMP). These structures, together with our recently published structure of the unligand human miniTyrRS (14), enabled us to achieve structure-based alignments that show a clear path for the development of Trp vs. Tyr discrimination.…”