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. In vitro using E. coli serRS and aspRS, the best compounds demonstrated IC50 values in the low nanomolar range, with a clear preference for cytosine
or N3-methyluracil over uracil. X-ray crystallographic structures
of K. pneumoniae serRS and T. thermophilus 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 E. coli 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.