Journal of Molecular Biology
Volume 304, Issue 5, 15 December 2000, Pages 983-994
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Regular Article
Macromolecular Assemblage of Aminoacyl-tRNA Synthetases: Quantitative Analysis of Protein-Protein Interactions and Mechanism of Complex Assembly,☆☆

https://doi.org/10.1006/jmbi.2000.4242Get rights and content

Abstract

The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches. This macromolecular assemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the seven monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 proteins. The scaffold p38 protein was expressed in Escherichia coli and purified to homogeneity as a His-tagged protein. The different components of the complex were shown to associate in vitro with p38 immobilized on Ni2+-coated plates. Interactions between peripheral enzymes and p38 are referred to as central interactions, as opposed to lateral interactions between peripheral enzymes. Kinetic parameters of the interactions were determined by the means of a biosensor-based approach. The two dimeric proteins LysRS and AspRS were found to tightly bind to p38, with a Kd value of 0.3 and 4.7 nM, respectively. These interactions involved the catalytic core of the enzymes. By contrast, binding of ArgRS or GlnRS to p38 was much weaker (>5 μM). ArgRS and p43, two peripheral components, were shown to interact with moderate affinity (Kd=93 nM). Since all the components of the complex are tightly associated within this particle, lateral interactions were believed to contribute to the stabilization of this assemblage. Using an in vitro binding assay, concomitant association of several components of the complex on immobilized p38 could be demonstrated, and revealed the involvement of synergistic effects for association of weakly interacting proteins. Taking into account the possible synergy between central and lateral contributions, a sub-complex containing p38, p43, ArgRS and GlnRS was reconstituted in vitro. These data provide compelling evidence for an ordered and concerted mechanism of complex assembly.

References (35)

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    The source of viral LysRS has been an active area of investigation. In higher eukaryotic cells, LysRS has been reported to be found in the nucleus (63), in mitochondria (64), assembled in the MSC in the cytosol (65), and associated with the plasma membrane (66, 67). Early studies suggested that newly synthesized LysRS is packaged into the virus prior to assembly into the MSC (66, 68).

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Abbreviations used: aminoacyl-tRNA synthetases are abbreviated by the three-letter code of the amino acid followed by RS (for instance, LysRS stands for lysyl-tRNA synthetase); Ni-NTA, nickel nitrilotriacetic acid; BSA, bovine serum albumin; SPR, surface plasmon resonance

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Edited by J. Karn

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Corresponding author

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E-mail address of the corresponding author: [email protected]

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