Our goal is to correlate protein conformational changes that take place during aminoacylation of tRNA by tryptophanyl-tRNA synthetase (TrpRS) from B stearothermophilus with the specific recognition and chemical transformations of the substrates. We plan to accomplish this by solving X-ray crystal structures representing additional stages of the catalytic cycle and comparing them with the now known structure of the trptophanyl- 5'AMP complex, whose structure analysis we will also extend to its diffraction limit of 1.7 A. The new crystal structures include a product complex containing tryptophanyl-2'3'-ATP, the Michaelis complexes with tryptophan and ATP, the ligand-free enzyme, and a complex with the cognate tRNA. All crystal forms diffract to better than 3.0 A and will be solved by molecular replacement. Quantitative analysis of crystal growth conditions, small angle X-ray scattering, and fluorescence studies have demonstrated that these crystal forms represent at least two and probably three different conformations. We will focus first on the product complex crystals whose ligand, trp- 2'(3')ATP, resembles the naturally occurring acyl-transfer product, tryptophanyl-tRNA/trp. That structure should provide insight into tRNA binding and hydroxyl group specificity of TrpRS. Then we will complete the structure of the two Michaelis complexes. Comparing these two structures with that of the adenylate and product complexes will elucidate all the important binding sites in the active site and suggest how they change and interact during activation and acyl-transfer. Mutational analysis will be used to test ideas suggested by the structures, including hypotheses already formulated about residues responsible for amino acid specificity. Specific recognition of tRNA and especially the coupling between anticodon binding and catalysis of acyl transfer will be addressed by preparing crystals and solving the structure of an enzyme:tRNA complex. Purified tRNA/trp is now available from a plasmid bearing the B. subtilis tRNA/trp gene. Crystals have previously been obtained and shown by electrophoresis to contain stoichiometrically equivalent amounts of both enzyme and tRNA/trp. Thus, it should now be possible to test specific predictions about the complex developed from the structure of the adenylate complex, and compare it with other structures along the catalytic path. Especially important will be to compare the complex with that previously obtained for GlnRS, and to explain how suppression with tryptophan occurs with an anticodon mutant of tRNA/gln. Together with the known structure, the new structures will constitute an extensive set of enzyme:ligand complexes, exceeding the range of structures previously available for other synthetases. They should therefore provide an unprecedented opportunity to examine how enzyme:ligand interactions and protein conformations change during catalysis of tryptophan activation and acyl-transfer, and hence how the correct amino acid and tRNA are selected.
| Carter Jr, Charles W (2017) Coding of Class I and II Aminoacyl-tRNA Synthetases. Adv Exp Med Biol 966:103-148 |
| Rodin, Andrei S; Rodin, Sergei N; Carter Jr, Charles W (2009) On primordial sense-antisense coding. J Mol Evol 69:555-67 |
| Laowanapiban, Poramaet; Kapustina, Maryna; Vonrhein, Clemens et al. (2009) Independent saturation of three TrpRS subsites generates a partially assembled state similar to those observed in molecular simulations. Proc Natl Acad Sci U S A 106:1790-5 |
| Retailleau, Pascal; Weinreb, Violetta; Hu, Mei et al. (2007) Crystal structure of tryptophanyl-tRNA synthetase complexed with adenosine-5'tetraphosphate: evidence for distributed use of catalytic binding energy in amino acid activation by class I aminoacyl-tRNA synthetases. J Mol Biol 369:108-28 |
| Kapustina, Maryna; Weinreb, Violetta; Li, Li et al. (2007) A conformational transition state accompanies tryptophan activation by B. stearothermophilus tryptophanyl-tRNA synthetase. Structure 15:1272-84 |
