Abstract
Although AIMP3/p18 is normally associated with the multi-tRNA synthetase complex via its specific interaction with methionyl-tRNA synthetase, it also works as a tumor suppressor by interacting with ATM, the upstream kinase of p53. To understand the molecular interactions of AIMP3 and the mechanisms involved, we determined the crystal structure of AIMP3 at 2.0-angstroms resolution and identified its potential sites of interaction with ATM. AIMP3 contains two distinct domains linked by a 7-amino acid (Lys57-Ser63) peptide, which contains a 3(10) helix. The 56-amino acid N-terminal domain consists of two helices into which three antiparallel beta strands are inserted, and the 111-amino acid C-terminal domain contains a bundle of five helices (Thr64-Tyr152) followed by a coiled region (Pro153-Leu169). Structural analyses revealed homologous proteins such as yeast glutamyl-tRNA synthetase, Arc1p, EF1Bgamma, and glutathione S-transferase and suggested two potential molecular binding sites. Moreover, mutations at the C-terminal putative binding site abolished the interaction between AIMP3 and ATM and the ability of AIMP3 to activate p53. Thus, this work identified the two potential molecular interaction sites of AIMP3 and determined the residues critical for its tumor-suppressive activity through the interaction with ATM.
Highlights
Two AIMP3 molecules (A and B) were [26], prostaglandin D synthase [27], yeast prion protein Ure2p found to be present in the asymmetric crystal unit and to be [28], E. coli stringent starvation A [29], and N-terminal related by a non-crystallographic 2-fold axis of symmetry. domains from yeast Arc1p and glutamyl-tRNA synthetase
Mutagenesis—We investigated the functions of AIMP3 residues that are important for its inter
Effect of Mutation on ATM Binding and p53 Activation—We introduced alanine substitutions to the residues of AIMP3 (Thr35/Ser40, Thr76, Thr80, Ser87, Val106, and Arg144) whose mutations have been identified in CML patients and examined whether any of these mutations affect its interactions with ATM and methionyltRNA synthetase (MRS) using in vitro pulldown and coimmunoprecipitation assays
Summary
Expression, and Purification of Human AIMP3— The cDNA encoding human AIMP3 was amplified by PCR and subcloned into pProEx-HTa (NdeI), a His fusion expression vector containing a recombinant tobacco etch virus protease cleavage site and a site for NdeI. SeMet-substituted AIMP3 (⌬C5,T42M/I70M/ K96M) was expressed in M9 minimal medium supplemented with 50 mg/ml SeMet. Cell pellets were resuspended in ice-cold buffer A (50 mM Tris-HCl, pH 8.0, 5 mM -mercaptoethanol) and disrupted by ultrasonication. The SeMet-substituted AIMP3 deletion mutant protein (human AIMP3⌬C5,T42M/I70M/K96M) was subjected to crystallization screening. Coimmunoprecipitation—Myc-tagged wild-type AIMP3 and mutants were transfected into 293 cells with the FLAG-tagged FAT domain of ATM. Luciferase Reporter Gene Assay—HCT116 p53ϩ/Ϫ cells were not defined because of poor electron density in both molecules grown in RPMI 1640 medium (HyClone) supplemented with A and B, suggesting a flexible loop connecting the two helices. Each of Average B-factors of molecules A and B are 31.7 and 33.4 Å2, the plasmids encoding the wild-type and mutant AIMP3 was respectively.
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