The use of wet lab and computational docking experiments to elucidate idiosyncrasies associated with amino acid activation

Abstract

Aminoacyl‐tRNA synthetases (aaRSs) are a well‐known class of enzymes that act as sentinels of genome. During protein translation, aaRSs ensure that L‐amino acids are charged or acylated onto their cognate tRNA isoacceptor(s). Hence, aaRSs help ensure that ribosomally translated proteins contain the proper sequence of amino acids. aaRS catalyze tRNA charging in a two‐step process. In step one, aaRSs selectively form a high energy amino acid adenylate. This step, refereed to amino acid activation, is the first level of selectivity in protein translation. Here, aaRSs effectively discriminate non‐cognate L‐amino acids. In step two, aaRSs transfer adenylated amino acids onto the cognate tRNA isoacceptor. This step is referred to as aminoacylation or tRNA charging. A great deal is known about aaRS reaction mechanisms but a number of confounding idiosyncrasies remain. The focus of this work aims to elucidate idiosyncrasies associated with amino acid activation. Here, a 32P‐based assay is used to measure the level to which E. coliaaRSs activate non‐cognate L‐amino acids. The purpose of these assays is to measure the plasticity of aaRS active sites to define additional underlying binding interactions that modulate amino acid recognition. This information could also be used to develop small molecule inhibitors of aaRS. Moreover, these data could reveal underlying clues to the potential origins of aaRSs. Here, three aaRSs are investigated: ArgRS, IleRS and LysRS. The activation data shows that each aaRS perhaps somewhat surprisingly activates more than five non‐cognate AAs to varying levels. Next, computer docking experiments are used to visualize the binding interaction of synthetase active site residues to each non‐cognate amino acid. These data are currently being used to construct protein‐ligand interaction maps of each aaRS active site. The resulting interactions maps can be used to more clearly elucidate idiosyncrasies associated with amino activation.