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.

FADD is known to function as a common signaling conduit in Fas-and tumor necrosis factor (TNF)-mediated apoptosis. The convergent death signals from the Fas receptor and TNF receptor 1 are transferred to FADD by death domain interactions triggering the same cellular event, caspase-8 activation. In this work, we investigated whether the same binding surface of FADD is used for both signaling pathways by using FADD death domain mutants. Mutations in helices ␣2 and ␣3 of the FADD death domain, the interacting surface with the Fas death domain, affected TNF-mediated apoptosis to various extents. This indicated that TNF-mediated apoptosis uses the same binding surface of the FADD death domain as Fas-mediated apoptosis. The binding specificity is not the same, however. Some mutations affected the binding affinity of the Fas death domain for the FADD death domain, but did not influence TNFmediated apoptosis and vice versa. Interestingly, all mutants tested that affected TNF-mediated apoptosis have structural perturbations, implying that the structural integrity, involving helices ␣2 and ␣3 in particular, is critical in TNF-mediated apoptosis. Our results suggest that different signaling molecules use a similar structural interaction to trigger the same cellular event, such as caspase-8 recruitment, which could be typical in convergent signal transduction.The tumor necrosis factor (TNF) receptor family consists of type I transmembrane proteins characterized by cysteine-rich repeats in their extracellular ligand-binding domains (1). A subset of the family, comprising CD95/Fas, TNFR1, p75 nerve growth factor receptor, DR3 (death receptor 3), and the two TRAIL (TNF-related apoptosis-inducing ligand) receptors (DR4/TRAIL receptor 1 and DR5/TRAIL receptor 2), shares a conserved motif in the cytoplasmic regions known as the death domain, which is essential for transducing the apoptotic signal (2-4). Upon receptor ligation, the death domain acts as the docking site for homotypic interaction with death domain-containing cytoplasmic proteins such as FADD (Fas-associated death domain protein) (5, 6) and TRADD (TNFR1-associated death domain protein) (7). FADD binds directly to Fas and is also recruited to TNFR1, DR3, and possibly other related receptors via TRADD (3,8). The death effector domain at the N-terminal end of FADD then interacts with a related motif in the prodomain of caspase-8 (5, 9) or caspase-10b (10). Activation of these upstream cysteine proteases is thought to trigger a proteolytic cascade, which apparently constitutes the execution of apoptosis. Under certain environments, however, Fas and other members of the TNFR family that contain death domains can stimulate alternative signaling pathways, which exert positive effects on cell survival and proliferation rather than triggering apoptosis (1).Endogenous FADD associates with Fas in an activation-dependent fashion (11), and TRADD and RIP (receptor-interacting protein) have been found in the activated TNFR1 complex (8). It has been postulated that TRADD acts as an ...

No abstract

No abstract