Abstract
The hypermodified nucleoside N(6)-threonylcarbamoyladenosine (t(6)A37) is present in many distinct tRNA species and has been found in organisms in all domains of life. This post-transcriptional modification enhances translation fidelity by stabilizing the anticodon/codon interaction in the ribosomal decoding site. The biosynthetic pathway of t(6)A37 is complex and not well understood. In bacteria, the following four proteins have been discovered to be both required and sufficient for t(6)A37 modification: TsaC, TsaD, TsaB, and TsaE. Of these, TsaC and TsaD are members of universally conserved protein families. Although TsaC has been shown to catalyze the formation of L-threonylcarbamoyl-AMP, a key intermediate in the biosynthesis of t(6)A37, the details of the enzymatic mechanism remain unsolved. Therefore, the solution structure of Escherichia coli TsaC was characterized by NMR to further study the interactions with ATP and L-threonine, both substrates of TsaC in the biosynthesis of L-threonylcarbamoyl-AMP. Several conserved amino acids were identified that create a hydrophobic binding pocket for the adenine of ATP. Additionally, two residues were found to interact with L-threonine. Both binding sites are located in a deep cavity at the center of the protein. Models derived from the NMR data and molecular modeling reveal several sites with considerable conformational flexibility in TsaC that may be important for L-threonine recognition, ATP activation, and/or protein/protein interactions. These observations further the understanding of the enzymatic reaction catalyzed by TsaC, a threonylcarbamoyl-AMP synthase, and provide structure-based insight into the mechanism of t(6)A37 biosynthesis.
Highlights
Threonylcarbamoyl-AMP synthase catalyzes formation of the biosynthetic intermediate of a critical transfer RNA (tRNA) modification, t6A37
We report the high-resolution structure of the monomer and interaction studies with L-threonine and ATP in solution using nuclear magnetic resonance (NMR)
NMR spectra of the protein in the absence of any ligands were the same collected under buffer conditions for structural determinations (90:10% H2O/2H2O, 20 mM potassium phosphate, pH 7.0, 100 mM NaCl) that were slightly different in pH and ionic strength from conditions for the protein’s titration with ATP and L-threonine followed by NMR and isothermal titration calorimetry (ITC) (90:10% H2O/2H2O, 50 mM potassium phosphate, pH 7.5, 150 mM KCl)
Summary
Threonylcarbamoyl-AMP synthase catalyzes formation of the biosynthetic intermediate of a critical tRNA modification, t6A37. The t6A37 modification has been shown to have an important role in ribosome-mediated codon binding for several tRNA species (6 – 8), mainly due to its ability to enhance the stability of the anticodon-codon base pairing by creating cross-strand basestacking interactions with the first position of the codon, as depicted in structural analyses of tRNALUyUsU decoding at the ribosome A-site [9, 10]. These studies provide insight into how TsaC catalyzes the formation of TC-AMP
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