Poster abstracts

Poster number 51 submitted by Sheng Liu

Structural studies of the tRNA binding domains of human lysyl-tRNA synthetase

Sheng Liu (Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172), Jason Jordan (Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172), Tekeya Waters (University of Maryland Eastern Shore, Princess Anne, MD 21853), Mike Howell (Protein Express, Cincinnati, OH 45241), Pearl Tsang (Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172)

Abstract:
Aminoacyl tRNA synthetases link tRNA molecules correctly to their cognate amino acids for protein biosynthesis. Human lysyl-tRNA synthetase (hKRS) has other functions including its secretion to trigger a proinflammatory response, and its interaction with Gag for uptake and packaging into HIV-1. In our research, NMR studies of the N terminal as well as the anticodon-binding (ACB) domains of hKRS are being studied individually as well as in tandem as a protein referred to here as ‘2D’. These studies are aimed at obtaining a better understanding of how these domains interact with the tRNA. For this purpose, 15N isotopically labeled forms of the N terminal domain, ACB domain and the 2D protein were expressed and purified for NMR study. Titrations of the N terminal, ACB as well as the 2D domains using RNA (oligo U or anticodon stem-loop) were monitored via a series of 1H-15N HSQC experiments. The shifting as well as broadening of numerous protein resonances was observed via RNA titration experiments of the ACB and 2D proteins reflecting the direct interaction of the RNA and ACB domains with each other. NMR studies of the N terminal domain protein indicate that is disordered by itself as well as when it is covalently attached to the ACB domain. The latter domain is much more highly structured by comparison. Further triple resonance experiments conducted upon doubly labeled ACB domain protein will provide site-specific information as far as which portions of this domain are most important for these specific interactions with the RNA. The overall picture of RNA binding by these domains will be presented and discussed.

Keywords: tRNA binding, human lysyl-tRNA synthetase, NMR