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Probing the Evolution of New Specificities in Aminoacyl-Trna Synthetases

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Probing the Evolution of New Specificities in Aminoacyl-Trna Synthetases Probing the Evolution of New Specificities in Aminoacyl-tRNA Synthetases Presented in Partial Fulfillment of the Requirements for the Master’s Degree in the Graduate School of The Ohio State University By Marla S. Gilreath, B.S. Graduate Program in Biochemistry The Ohio State University 2011 Master’s Committee: Dr. Venkat Gopalan, Advisor Dr. Michael Ibba Copyright by Marla S. Gilreath 2011 ABSTRACT Bacterial elongation factor P (EF-P) is a poorly understood soluble protein that has been shown to enhance the first step of peptide bond formation through an interaction with the ribosome and initiator tRNA. Homologous proteins have been found in both archaeal and eukaryotic systems, known as aIF5A and eIF5A, respectively. eIF5A, which was recently shown to increase translation elongation rates, is post-translationally modified at a highly conserved lysine residue through the addition of the rare amino acid hypusine. A similar pathway was recently elucidated for EF-P, in which EF-P is post- translationally modified by the enzymes PoxA and YjeK at lysine 34, corresponding to a homologous site of hypusination in a/eIF5A. As a paralog of class II LysRS, PoxA catalyzes the addition of lysine onto EF-P, but is incapable of modifying tRNA. YjeK is a 2,3-(β)-lysine aminomutase and is responsible for converting lysine to β-lysine, which PoxA was recently shown to recognize as a preferred substrate for EF-P modification. The amino acid binding pockets of LysRS and PoxA are highly conserved, with the exception of two residues, Gly465 and Ala229 of Geobacillus stearothermophilus LysRS and Ala298 and Ser76 of Salmonella Typhimurium PoxA. Despite their substantial active site similarity, PoxA exhibits a significantly higher KM value for activation of lysine as compared to LysRS. This suggests that the two divergent residues in the active site determine the specificity for substrate recognition and binding, as well ii as optimal enzymatic activity, of PoxA. To investigate the mechanisms of α- versus β- amino acid recognition in the divergent evolution of LysRS and PoxA, three amino acid replacements were made in the LysRS active site. Kinetic parameters for ATP/PPi exchange reactions were determined for wild type, A233S, and G469A LysRS and aminoacylation reactions were carried out to further characterize the activity of each variant. Results indicate that while A233S behaves like the wild type, G469A and A233S/G469A significantly decrease the ability of LysRS to form stable lysyl- adenylates. A233S was able to shift the substrate specificity of LysRS to recognize (S)-β- lysine, indicating that few active-site substitutions are necessary to facilitate changes in the substrate specificity of an aaRS. iii ACKNOWLEDGEMENTS I would like to extend a sincere thank you to Dr. Michael Ibba for fostering my graduate education in biochemistry and expanding my knowledge of experimental techniques. I am truly grateful for the time I spent learning from Dr. Ibba and my fellow Ibba Lab members, in particular Dr. Hervé Roy, who patiently facilitated my training as a protein biochemist. I would also like to thank Dr. Venkat Gopalan for holding the position as my advisor of record. iv VITA June 2004 .......................................................Cumberland Valley High School May 2008 .......................................................B.S. Biochemistry, University of Delaware 2008 to present ..............................................Graduate Research Assistant, Department of Microbiology, The Ohio State University PUBLICATIONS Roy, H., Zou, S.B., Bullwinkle, T., Wolfe, B., Gilreath, M., Forsyth, C., Navarre, W.W., and Ibba, M. The tRNA synthetase paralog PoxA modifies elongation factor P with (R)-β-lysine. Nature Chemical Biology (In press) Banerjee, R., Chen, S., Dare, K., Gilreath, M., Praetorius-Ibba, M., Raina, M., Reynolds, N., Rogers, T.E., Roy, H., Yadavalli, S.S., and Ibba, M. (2009) tRNA: Cellular barcodes for amino Acids. FEBS Lett. 584: 387-395. FIELDS OF STUDY Major Field: Biochemistry v TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii ACKNOWLEDGEMENTS............................................................................................... iv VITA................................................................................................................................... v PUBLICATIONS................................................................................................................ v FIELDS OF STUDY .......................................................................................................... v TABLE OF CONTENTS................................................................................................... vi LIST OF TABLES............................................................................................................ vii LIST OF FIGURES .........................................................................................................viii INTRODUCTION .............................................................................................................. 1 MATERIALS AND METHODS...................................................................................... 26 RESULTS ......................................................................................................................... 30 DISCUSSION................................................................................................................... 38 OUTLOOK ....................................................................................................................... 41 REFERENCES ................................................................................................................. 45 vi LIST OF TABLES Table 1. Concentrations of purified proteins…………………………………………….31 Table 2. Steady-state kinetics of lysine activation……………………………………….34 vii LIST OF FIGURES Figure 1. aaRS-catalyzed aminoacylation reaction............................................................. 2 Figure 2. Aminoacylation and co-translational insertion of an amino acid........................ 3 Figure 3. Overview of mRNA-encoded protein synthesis.................................................. 5 Figure 4. Structural comparison of EF-P with tRNA and other ribosome-binding proteins ............................................................................................................................................. 8 Figure 5. Crystal structure of EF-P bound to the 70S ribosome....................................... 12 Figure 7. Proteins aIF5a and eIF5A from M. jannaschii and L. mexicana....................... 15 Figure 8. Structural comparison of EF-P and eIF5A ........................................................ 16 Figure 9. Post-translational modification pathways of eIF5A and EF-P.......................... 17 Figure 10. Current model for the post-translational modification of EF-P....................... 19 Figure 11. PoxA-catalyzed in vitro lysylation of EF-P..................................................... 20 Figure 12. Molecular docking model comparison of LysRS and PoxA active sites. ....... 21 Figure 13. Aminoacylation of LysRS wild type and variants........................................... 32 Figure 14. Representative Michaelis-Menten curves for wild type (A), G469 (B), and A233S (C) ......................................................................................................................... 33 Figure 15. Amino acid specificity of LysRS wild type and A233S with (S)-β-lysine and (R)-β-lysine ....................................................................................................................... 36 viii INTRODUCTION mRNA-encoded Protein Synthesis First posed as the “coding problem” by biologists in the late 1950s, translation involves the conversion of genetic information from DNA to protein by following a set of rules deemed the genetic code. During translation, the process of protein synthesis, nucleotides in a messenger RNA (mRNA) are decoded to direct the incorporation of amino acids into a growing peptide chain. Each set of three consecutive nucleotides, referred to as codons, either directs the incorporation of a specific amino acid or signals a stop to translation. At least one codon represents each of the 20 amino acids, with multiple codons existing for the more frequently used amino acids. The same genetic code is universally employed by all organisms for protein synthesis, although some differences do occur, for example, in the DNA of mitochondria and in other rare instances (1). The accurate translation of mRNA to protein requires the assistance of adaptor molecules capable of binding to both the mRNA codon and the cognate amino acid to be incorporated. These substrates for translation are termed aminoacyl-tRNAs (aa-tRNAs), and there exists one tRNA with the correct anticodon region for each amino acid of the genetic code. The 20 aa-tRNAs can be divided into three categories: translation substrates 1 (canonical elongator aa-tRNAs, initiator aa-tRNAs, or non-canonical aa-tRNAs), misacylated translation substrates, or nontranslation substrates (2). Cognate amino acids are esterified onto the 3’-end of the mature transfer RNA (tRNA) through a reaction catalyzed by aminoacyl-tRNA synthetases (aaRSs). This aminoacylation reaction occurs in two steps: first the amino acid is activated with ATP to form an aminoacyl-adenylate (aaAMP), and second the activated
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