Structural Insights into the Binding of Natural Pyrimidine-Based Inhibitors of Class II Aminoacyl-tRNA Synthetases.

The pyrimidine-containing Trojan horse antibiotics albomycin and a recently discovered cytidine-containing microcin C analog target the class II seryl- and aspartyl-tRNA synthetases (serRS and aspRS), respectively. The active components of these compounds are competitive inhibitors that mimic the aminoacyl-adenylate intermediate. How they effectively substitute for the interactions mediated by the canonical purine group is unknown. Employing nonhydrolyzable aminoacyl-sulfamoyl nucleosides substituting the base with cytosine, uracil, and N3-methyluracil the structure-activity relationship of the natural compounds was evaluated. using serRS and aspRS, the best compounds demonstrated IC values in the low nanomolar range, with a clear preference for cytosine or N3-methyluracil over uracil. X-ray crystallographic structures of serRS and aspRS in complex with the compounds showed the contribution of structured waters and residues in the conserved motif-2 loop in defining base preference. Utilizing the N3-methyluracil bound serRS structure, MD simulations of the fully modified albomycin base were performed to identify the interacting network that drives stable association. This analysis pointed to key interactions with a methionine in the motif-2 loop. Interestingly, this residue is mutated to a glycine in a second serRS (serRS2) found in albomycin-producing actinobacteria possessing self-immunity to this antibiotic. A comparative study demonstrated that serRS2 is poorly inhibited by the pyrimidine-containing intermediate analogs, and an equivalent mutation in serRS significantly decreased the affinity of the cytosine congener. These findings highlight the crucial role of dynamics and solvation of the motif-2 loop in modulating the binding of the natural antibiotics.