Poster abstracts

Poster number 3 submitted by Jeffrey Levengood

Rationale for the evolutionary retention of 2 unrelated LysRSs

Jeffrey Levengood (Department of Microbiology, The Ohio State University), Sandro Ataide (Department of Microbiology, The Ohio State University), Osamu Nureki (Department of Biophysics and Biochemistry, University of Tokyo), Michael Ibba (Deparmtent of Microbiology, The Ohio State University)

Abstract:
Lysyl-tRNALys can be synthesized by a Lysyl-tRNA synthetase (LysRS) belonging to either synthetase class I (LysRS1), or class II (LysRS2). LysRS is the only synthetase known to violate the class rule, leading to questions as to why nature selected two different enzymes for performing the same function. The solving of the crystal structure of LysRS1 from Pyrococcus horikoshii gave some insight to this question. The structure solved with and without L-lysine revealed the composition of the lysine binding pocket. Comparison with the structure of LysRS2 from E. coli showed the LysRS1 binding pocket to be more compact than the one for LysRS2. In vitro analysis with L-lysine analogues supported this assertion as the Kis for S-(2-aminoethyl)-L-cysteine and L-lysinamide were at least 180 fold lower for LysRS2 than LysRS1. In contrast, gamma-aminobutyric acid was found to have a much lower Ki for LysRS1 than LysRS2. These differences were confirmed through in vivo experiments.
The LysRS1 crystal structure revealed aspects of the way tRNA binds with the enzyme. The anticodon loop of the tRNA is recognized by an alpha-helix cage domain that is only found in one other synthetase. While the structure could not be solved with tRNA bound to the enzyme, modeling was done to determine how the enzyme might recognize the tRNA. From this model amino acids that bind the anticodon nucleotides on tRNALys were identified. Variant enzymes were then created along with tRNALys mutants that contain different anticodon nucleotides. Kinetic analysis was done by finding the kcat/KM for each enzyme-tRNA combination. The intent of this project is to combine the biochemical data with energy minimization modeling to build a new docking mock-up. Discovering how LysRS1 binds the anticodon loop of its tRNA could provide interesting contrast when compared to the better studied OB-fold domain of LysRS2.

References:
Jester, B., Levengood, J., Roy, H., Ibba, M., and DeVine, K. (2003) Proc. Natl. Acad. Sci. USA 10014351-14356.
Levengood, J., Ataide, S., Roy, H., and Ibba, M. (2004) J. Biol. Chem. 279:17707-17714.
Terada, T., Nureki, O., Ishitani, R., Ambrogelly, A., Ibba, M., Söll, D., and Yokoyama, S. (2002) Nat. Struct. Biol. 9(4):257-262.

Keywords: synthetase, tRNA