Cladosporin (CLD) is a fungal metabolite that kills the
malaria parasite via inhibiting its cytoplasmic
lysyl-tRNA synthetase (KRS) and abrogating protein translation. Here we provide structural and
drug selectivity analyses on CLD interacting residues in apo and holo KRSs from Plasmodium falciparum, Homo sapiens, Cryptosporidium parvum, and Mycobacterium ulcerans. We show that both gross and subtle alterations in
protein backbone and sidechains drive the active site structural plasticity that allows integration of CLD in KRSs. The
ligand-induced fit of CLD in PfKRS is marked by closure and stabilization of three disordered loops and
one alpha helix. However, these structural rearragements are not evident in KRS-CLD complexes from H. sapiens, C. parvum, or M. ulcerans. Strikingly, CLD fits into the MuKRS active site due to a remarkable rotameric alteration in its clash-prone
methionine residue that provides accommodation for the methyl moiety in CLD. Although the high concentrations of drugs used for Hs, Cp, and MuKRS-CLD complexes in co-crystallization studies enable elucidation of a structural framework for understanding
drug binding in KRSs, we propose that these data should be concurrently assessed via biochemical studies of potency and
drug selectivity given the poor cell-based activity of CLD against human and bacterial cells. Our comprehensive analyses of KRS-CLD interactions, therefore, highlight vital issues in structure-based
drug discovery studies.