Identification of protein interfaces within the multi‐aminoacyl‐tRNA synthetase complex: the case of lysyl‐tRNA synthetase and the scaffold protein p38

Abstract

Human cytoplasmic lysyl‐ tRNA synthetase (Lys RS ) is associated within a multi‐aminoacyl‐ tRNA synthetase complex ( MSC ). Within this complex, the p38 component is the scaffold protein that binds the catalytic domain of Lys RS via its N‐terminal region. In addition to its translational function when associated to the MSC , Lys RS is also recruited in nontranslational roles after dissociation from the MSC . The balance between its MSC ‐associated and MSC ‐dissociated states is essential to regulate the functions of Lys RS in cellular homeostasis. With the aim of understanding the rules that govern association of Lys RS in the MSC , we analyzed the protein interfaces between Lys RS and the full‐length version of p38, the scaffold protein of the MSC . In a previous study, the cocrystal structure of Lys RS with a N‐terminal peptide of p38 was reported [Ofir‐Birin Y et al . (2013) Mol Cell 49, 30–42]. In order to identify amino acid residues involved in interaction of the two proteins, the non‐natural, photo‐cross‐linkable amino acid p ‐benzoyl‐ l ‐phenylalanine (Bpa) was incorporated at 27 discrete positions within the catalytic domain of Lys RS . Among the 27 distinct Lys RS mutants, only those with Bpa inserted in place of Lys356 or His364 were cross‐linked with p38. Using mass spectrometry, we unambiguously identified the protein interface of the cross‐linked complex and showed that Lys356 and His364 of Lys RS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in Lys RS is located on the opposite side of the dimer to the site of interaction with its tRNA substrate, defines the core region of the MSC . The residues identified herein in human Lys RS are not conserved in yeast Lys RS , an enzyme that does not associate within the MSC , and contrast with the residues proposed to be essential for Lys RS :p38 association in the earlier work.

Characterization of association of human mitochondrial lysyl-tRNA synthetase with HIV-1 Pol and tRNA3Lys

“…In human, cLysRS associates with 8 other aminoacyl-tRNA synthetases and with the 3 auxilliary proteins p18, p38 and p43 to form the multi-synthetase complex (MSC), which is a strictly cytoplasmic entity [ 13 ]. Its association is mediated by the scaffold protein p38, also named AIMP2 [ 14 , 15 ].…”

Mutations in RARS cause a hypomyelination disorder akin to Pelizaeus–Merzbacher disease

“…These ARS proteins along with three ARS-interacting multifunctional proteins namely p18, p38, and p43, are organised into a macromolecular protein complex called multi-synthetase complex (MSC). 24 The MSC, has an estimated molecular weight of 1.5 MDa and is involved in protein synthesis and diverse cellular signalling pathways. 25,26 There are two isoforms of cytoplasmic RARS in human cells.…”

How HIV-1 Integrase Associates with Human Mitochondrial Lysyl-tRNA Synthetase

“…To identify the surface area of mLysRS involved in its interaction with the CTD of integrase from HIV-1, 35 variants of mLysRS carrying each a single p Bpa inserted at the surface of the protein and evenly distributed at the surface of the catalytic domain ( Table S2 ) were isolated as described previously [ 24 ]. The wild-type and p Bpa-containing mLysRS proteins were incubated on ice at a dimer concentration of 85 nM in the presence of IN-CTD222-H 6 at a monomer concentration of 1.12 µM, in PBS buffer.…”

“…The QuickChange Lightning Site-directed Mutagenesis Kit from Agilent Technologies was used to introduce amber (TAG) stop codons at discrete sites within the nucleotide sequence encoding the catalytic domain of mLysRS or the CTD222 domain of integrase from HIV-1 as described previously [ 24 ]. Incorporation of p -benzoyl-L-phenylalanine ( p Bpa) into mutant proteins was conducted in E. coli BL21(DE3) transformed with pEVOL-Bpa that expresses the orthologous supression system [ 25 ].…”