Macromolecular assemblage of aminoacyl-tRNA synthetases: Quantitative analysis of protein-protein interactions and mechanism of complex assembly

The structure of the mammalian multi-synthetase complex was investigated in vitro using qualitative and quantitative approaches. This macromolecular a ssemblage comprises the bifunctional glutamyl-prolyl-tRNA synthetase, the s even monospecific isoleucyl, leucyl, methionyl, glutaminyl, lysyl, arginyl and aspartyl-tRNA synthetases, and the three auxiliary p43, p38 and p18 pro teins. The scaffold p38 protein was expressed in Escherichia coil and purif ied to homogeneity as a His-tagged protein. The different components of the complex were shown to associate in vitro with p38 immobilized on Ni2+-coat ed plates. Interactions between peripheral enzymes and p38 are referred to as central interactions, as opposed to lateral interactions between periphe ral enzymes. Kinetic parameters of the interactions were determined by the means of a biosensor-based approach. The two dimeric proteins LysRS and Asp RS were found to tightly bind to p38, with a K-d value of 0.3 and 4.7 nM, r espectively. These interactions involved the catalytic core of the enzymes. By contrast, binding of ArgRS or GlnRS to p38 was much weaker (>5 muM). Ar gRS and p43, two peripheral components, were shown to interact with moderat e affinity (K-d = 93 nM). Since all the components of the complex are tight ly associated within this particle, lateral interactions were believed to c ontribute to the stabilization of this assemblage. Using an in vitro bindin g assay, concomitant association of several components of the complex on im mobilized p38 could be demonstrated, and revealed the involvement of synerg istic effects for association of weakly interacting proteins. Taking into a ccount the possible synergy between central and lateral contributions, a su b-complex containing p38, p43, ArgRS and GlnRS was reconstituted in vitro. These data provide compelling evidence for an ordered and concerted mechani sm of complex assembly. (C) 2000 Academic Press.