Poster abstracts

Poster number 62 submitted by Sheng Liu

Progress towards structural study of the N-terminal domain of human lysyl aminoacyl tRNA synthetase

Sheng Liu (Department of Chemistry, University of Cincinnati), Amaleah Hartman (Department of Chemistry, University of Cincinnati), Christopher Jones (Departments of Chemistry and Biochemistry, The Ohio State University), Karin Musier-Forsyth (Departments of Chemistry and Biochemistry, The Ohio State University), Mike Howell (ProteinExpress), Pearl Tsang (Department of Chemistry, University of Cincinnati)

Abstract:
Human lysyl aminoacyl tRNA synthetase (LysRS) is an essential protein biosynthesis enzyme that has a wide range of other, auxiliary functions ranging from pro-inflammatory response to chaperoning of the HIV-1 reverse transcription primer molecule, tRNA^Lys,3, during packaging of new HIV-1 particles (1, 2). An important characteristic of higher eukaryotic forms of LysRS is the appendage of an N-terminal domain to the highly conserved anticodon binding (ACB) and catalytic domains. The function of this N-terminal domain has been proposed to enhance the overall RNA binding affinity which may contribute to the HIV-1 reverse transcription primer uptake and possibly modulate LysRS binding to other proteins (3, 4, 5); however, its structure still remains unknown. Here, we present preliminary results of our NMR study to better elucidate the role and potential structure of this appendage domain. Based upon the assigned frequencies of the NMR resonances of this protein, the isolated N-terminal domain is mostly unstructured in solution, in contrast to its secondary structure that is predicted based upon its amino acid sequence. However, this domain does adopt a 27 residue long helical structure but only upon binding to a RNA ligand corresponding to the anticodon stem-loop of the cognate human tRNA^Lys,3. Furthermore, fluorescence anisotropy and EMSA binding studies show that the N-terminal domain of human LysRS does indeed enhance the overall RNA binding affinity of the ACB domain. NMR titration studies using different forms of the N-terminal domain protein via segmental labeling (6) reveal that RNA binding by this N-terminal domain is cooperative with the adjacent ACB domain. Potential RNA binding site of the N-terminal domain is also discussed.

References:
Reference:
1) Park SG, Kim HJ, Min YH, Choi E-C, Shin YK, Park B-J, Lee SW and Kim S (2005) PNAS 102(18):6356-6361
2) Cen S, Khorchid A, Javanbakht H, Gabor J, Stello T, Shiba K, Musier-Forsyth K and Kleiman L (2001) Journal of Virology 75:5043-5048
3) Francin M, Kaminska M, Kerjan P and Mirande M (2002) The Journal of Biological Chemistry 277:1762-1769
4) Cen S, Javanbakht H, Niu M and Kleiman L (2004) Journal of Virology 78:1595-1601
5) Guzzo C, Yang D (2008) Biochemical and Biophysical Research Communications 365:718–723
6) Refaei M, Combs A, Kojetin D, Cavanagh J, Caperelli C, Rance M, Sapitro J and Tsang P (2011) Journal of Biomolecular NMR 49:3-7

Keywords: LysRS, HIV-1, RNA-protein complex