Project Summary/Abstract Mammalian cells contain a cytoplasmic multi-tRNA synthetase complex (MSC) consisting of 8 aminoacyl-tRNA synthetases (AARSs) and 3 non-synthetase proteins. AARSs in the MSC function as “gene decoders” during mRNA translation, but also exhibit non-canonical functions outside the MSC. However, the assembly, structure, and function of the MSC are poorly understood. Importantly, mutations in genes encoding 7/11 constituents cause central nervous system (CNS) disorders – five cause hypomyelinating leukodystrophy (HLD), and two others cause progressive microcephaly. We will utilize state-of-the-art molecular approaches to improve our understanding of the MSC, and its potential role in neuropathology. Our proposed Multiple-PI program takes advantage of the expertise of two highly collaborative PI's – Paul Fox (Contact PI), a molecular biologist with long-term interest in tRNA synthetases and the MSC, and Valentin Gogonea (Multiple PI), a physical chemist with expertise in analysis and molecular modeling of multi-protein complexes. We will determine the quaternary structure of the MSC by cross-linking mass spectrometry (XL-MS), a state-of-the-art method that facilitates analysis of otherwise intractable complexes. To date we have found 19 inter-protein cross-links between all 11 MSC constituents, and 118 intra-protein cross-links. We have generated an initial model of the MSC that will be refined here by XL-MS experiments with expanded amino acid specificity, and by SiMPull (single-molecule pulldown) coupled with single-molecule fluorescence to determine stoichiometry. In addition, we will investigate the mechanism of assembly of the MSC. Constitutive, multi-protein complexes are thought to be assembled by domain-specific interactions between fully-formed, mature constituents (“post-translational assembly”). However, assembly of some complexes utilizes a “co-translational assembly” mechanism in which a mature constituent interacts with the nascent peptide of a partner constituent as it emerges from the ribosome. In preliminary data we show at least 10 pairs of MSC constituents interact co-translationally. We will apply these mechanistic approaches to elucidate the role of two MSC constituents in CNS diseases – genetic defects in QARS1 and EPRS1 that cause microcephaly and HLD, respectively. Our preliminary studies indicate that constituent mutation or suppression can lead to extra-MSC accumulation. Our preliminary studies have led us to propose the following hypothesis: The mammalian MSC is a compact structure assembled in part by an orderly sequence of co-translational interactions, however, mis-assembly or mutation can induce extra-MSC accumulation of constituents, with potentially deleterious downstream consequences. We will test this hypothesis by (1) determining MSC quaternary structure and component stoichiometry, and (2) determining the role of co-translational interactions in MSC formation and integrity. We anticipate tha...