Structural analysis of multifunctional peptide motifs in human bifunctional tRNA synthetase: Identification of RNA-binding residues and functional implications for tandem repeats

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three ta ndem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid inter actions. The single copy of the homologous motifs has also been found in se veral different aminoacyl-tRNA synthetases. The solution structure of repea t 1 (EPRS-R1) and the secondary structure of the whole appended domain cont aining three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (res idues 679-699 and 702-721) arranged in a helix-turn-helix, which is similar to other RNA binding proteins and the j-domain of DnaJ, and EPRS-R123 is c omposed of three helix-turn-helix motifs linked by an unstructured loop. Wh en tRNA is bound to the appended domain, chemical shifts of several residue s in each repeat are perturbed. However, the perturbed residues in each rep eat are not the same although they are in the same binding surface, suggest ing that each repeat in the appended domain is dynamically arranged to maxi mize contacts with tRNA. The affinity of tRNA to the three-repeated motif w as much higher than to the single motif. These results indicate that each o f the repeated motifs has a weak intrinsic affinity for tRNA, but the repet ition of the motifs may be required to enhance binding affinity. Thus, the results of this work gave information on the RNA-binding mode of the multif unctional peptide motif attached to different ARSs and the functional reaso n for the repetition of this motif.