Structural Analysis of Multifunctional Peptide Motifs in Human Bifunctional tRNA Synthetase:  Identification of RNA-Binding Residues and Functional Implications for Tandem Repeats^,

Abstract

Human bifunctional glutamyl-prolyl-tRNA synthetase (EPRS) contains three tandem repeats linking the two catalytic domains. These repeated motifs have been shown to be involved in protein-protein and protein-nucleic acid interactions. The single copy of the homologous motifs has also been found in several different aminoacyl-tRNA synthetases. The solution structure of repeat 1 (EPRS-R1) and the secondary structure of the whole appended domain containing three repeated motifs in EPRS (EPRS-R123) was determined by nuclear magnetic resonance (NMR) spectroscopy. EPRS-R1 consists of two helices (residues 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 composed of three helix-turn-helix motifs linked by an unstructured loop. When tRNA is bound to the appended domain, chemical shifts of several residues in each repeat are perturbed. However, the perturbed residues in each repeat are not the same although they are in the same binding surface, suggesting that each repeat in the appended domain is dynamically arranged to maximize contacts with tRNA. The affinity of tRNA to the three-repeated motif was much higher than to the single motif. These results indicate that each of the repeated motifs has a weak intrinsic affinity for tRNA, but the repetition 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 multifunctional peptide motif attached to different ARSs and the functional reason for the repetition of this motif.

Determination of Three-dimensional Structure and Residues of the Novel Tumor Suppressor AIMP3/p18 Required for the Interaction with ATM

“…The ␤ sheet was flanked by the parallel helices ␣1 and ␣2 on one side and exposed to solvent on the other side. The linker of 7 residues (Lys 57 -Ser 63 ), in which the peptide from Glu 58 to Leu 61 forms a 3 10 helix, connects the two domains. A short-turn helix is also found in the linker connecting two domains in the structures of GSTs from maize (22) and mosquito (23).…”

“…Arc1p has an ␣ helix ahead of the helical domain, which corresponds to the C-terminal domain of GST, and this first helix is apart from the second helix and is located near the fourth helix-like helix ␣1 in AIMP3-N. It also has a similar linker region containing a 3 10 helix between the first helix and the helical domain.…”

“…To understand the multifunctionality and regulation of the components of the multi-ARS complex, it is important to determine the three-dimensional structures of the components as well as the whole complex. So far, only the three-dimensional structures of the partial domains of the complex-forming ARSs such as glutamylprolyl-tRNA synthetase (10,11), aspartyltRNA synthetase (12), and AIMP1/p43 (13,14) have been elucidated. Here, we report the crystal structure of full-length AIMP3 and the residues and location required for the interaction with ATM.…”

A Three-dimensional Working Model of the Multienzyme Complex of Aminoacyl-tRNA Synthetases Based on Electron Microscopic Placements of tRNA and Proteins

“…However, GluProRS is a bifunctional polypeptide with two tRNA binding sites, which are clearly in close proximity. Moreover, the protein has a domain between the two active sites that has the property of general tRNA binding (27). Thus, two out of the three possible tRNA binding sites within GluProRS were likely to be occupied with sufficient frequency to produce the dual spots comprising site 2 (Fig.…”

Small interfering RNA knockdown of mini-TyrRS and mini-TrpRS effects angiogenesis in human umbilical vein endothelial cells in hypoxic culture

“…Mini-TyrRS has an ELR motif and promotes angiogenesis, but miniTrpRS does not have the ELR motif. Therefore, their function was totally opposite (Jeong et al 2000).…”