title: 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 (LysRS) 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 LysRS via its N-terminal region. In addition to its translational function when associated to the MSC, LysRS 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 LysRS in cellular homeostasis. With the aim of understanding the rules that govern association of LysRS in the MSC, we analyzed the protein interfaces between LysRS and the full-length version of p38, the scaffold protein of the MSC. In a previous study, the cocrystal structure of LysRS 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 LysRS. Among the 27 distinct LysRS 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 LysRS interact with the peptide from Pro8 to Arg26 in native p38, in agreement with the published cocrystal structure. This interface, which in LysRS 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 LysRS are not conserved in yeast LysRS, an enzyme that does not associate within the MSC, and contrast with the residues proposed to be essential for LysRS:p38 association in the earlier work.
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales (LEBS) CNRS Gif-sur-Yvette France.
authors.forename: Azaria
authors.initials: A
authors.lastname: Rémion
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales (LEBS) CNRS Gif-sur-Yvette France; Institute for Integrative Biology of the Cell (I2BC) CEACNRS Univ. Paris-Sud, Université Paris-Saclay Gif-sur-Yvette France.
authors.forename: Fawzi
authors.initials: F
authors.lastname: Khoder-Agha
authors.affiliation: Service d'identification et de Caractérisation des Protéines par Spectrométrie de Masse (SICaPS) CEA CNRS Univ. Paris-Sud, Université Paris-Saclay Gif-sur-Yvette France.
authors.forename: David
authors.initials: D
authors.lastname: Cornu
authors.affiliation: Service d'identification et de Caractérisation des Protéines par Spectrométrie de Masse (SICaPS) CEA CNRS Univ. Paris-Sud, Université Paris-Saclay Gif-sur-Yvette France.
authors.forename: Manuela
authors.initials: M
authors.lastname: Argentini
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales (LEBS) CNRSGif-sur-Yvette France; Service d'identification et de Caractérisation des Protéines par Spectrométrie de Masse (SICaPS) CEACNRS Univ. Paris-Sud, Université Paris-Saclay Gif-sur-Yvette France; Present address: Paris-Saclay Institute of Neuroscience (Neuro-PSI) CNRS 1 avenue de la Terrasse 91190 Gif-sur-Yvette France.
authors.forename: Virginie
authors.initials: V
authors.lastname: Redeker
authors.affiliation: Laboratoire d'Enzymologie et Biochimie Structurales (LEBS) CNRS Gif-sur-Yvette France; Institute for Integrative Biology of the Cell (I2BC) CEACNRS Univ. Paris-Sud, Université Paris-Saclay Gif-sur-Yvette France.
authors.forename: Marc
authors.initials: M
authors.lastname: Mirande
keywords: cross‐link; lysyl‐tRNA synthetase; multisynthetase complex; p38; protein:protein interaction