Author:

Keywords:

Aminoacyl-tRNA synthetase, Half-of-the-sites activity, X-ray crystallography, Structural Biology

Abstract:

Half-of-the-sites activity in the dimeric class I tyrosyl-tRNA synthetase (tyrRS) was reported more than four decades ago. Yet, despite a plethora of available structures, an understanding of the molecular mechanisms that result in this behavior is severely lacking. We have identified and optimized a new crystallization condition for the full-length E. coli tyrRS, where the resultant crystals routinely diffract beyond 2 Å resolution. The solved apo structure contains the canonical tyrRS dimer within the crystallographic asymmetric unit, where the two component subunits show significantly different conformations of conserved structural elements surrounding the active site. Crucially, either substrate or an analog of the reaction intermediate can be titrated into the obtained crystals by soaking. The resulting structures highlight the preferential binding of tyrosine to only one monomer and provide high-resolution snapshots of the structural transitions that occur upon association. To validate the “in crystallo” work we have performed additional solution studies using small-angle X-ray scattering and isothermal titration calorimetry, as well as characterization of tyrRS in the gas phase using native mass spectrometry. The combined efforts have permitted the development of a detailed model of tyrRS asymmetric catalytic activity. These results will be presented, and placed in context of the whole aaRS family, using comparative data we have obtained for other class I and class II representatives.