Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes that catalyze the first step of protein biosynthesis or translation. In the presence of ATP, they activate the amino acids as adenylates and subsequently bind the amino acid moity onto the 3' CCA end of transfer RNAs [1]. The resulting aminoacyl-tRNAs are then carried by the elongation factors to the ribosome to be incorporated into nascent polypeptide chains. AaRSs are a target of choice for drug design because they are essential enzymes having a high specificity for their substrates.
Our study is focused on bacterial aspartyl-tRNA synthetases (AspRSs) that bind specifically L-aspartate. We have initiated the structural characterization of the binding mode of two families of inhibitors. One is a natural antibiotic produced by E. coli strains that targets the catalytic site of AspRSs (collaboration with Prof. S. Rebuffat, Museum National d'Histoire Naturelle, Paris) and the other a series of chemically synthesized peptides that were selected against an AspRS from the opportunistic human pathogen Pseudomonas aeruginosa (collaboration with Prof. Hiroaki Suga, University of Tokyo).
We apply various crystallogenesis approaches to prepare crystals that are suitable for the X-ray diffraction analysis. They involve the optimization of crystal production either by cocrystallization or by soaking of native crystals with ligands. In the case where the enzyme of one bacterial species does not yield exploitable crystals, the protein is either chemically methylated to change its surface properties and crystallizability, or a close structural homolog with a conserved active site is substituted to take advantage of genetic diversity. In the final step crystallization systematically takes place in an agarose gel with a low gelling temperature to improve crystal quality, stability during the soaking with inhibitors, and handling [2]. The rationale of our crystallogenesis strategy will be presented and illustrated with examples.