Fig 1.
Domain structure, purification and activity of L. loa and S. mansoni KRSs.
(A) Domain-wise architectures of LlKRS and SmKRS are shown. SP, ABD and AAD denote signal peptide (mitochondrial), anticodon binding domain and aminoacylation domains respectively. (B) GPC elution profile of purified LlKRS (blue), SmKRS (red) with PfKRS (green). Comparison with standard markers shows that LlKRS and SmKRS elute at a size corresponding to dimeric states. No absorbance at tetrameric size was observed for either protein. (C) Final purified proteins on SDS-PAGE. LlKRSmut denotes the human-like LlKRS. (D) Time-dependent enzymatic activity assay for LlKRS, human-like LlKRS and SmKRS proteins at constant substrate concentrations show that purified enzymes were active for aminoacylation.
Fig 2.
Cladosporin activity on worm KRSs.
(A) Chemical structure of cladosporin. Cladosporin (CLD) is composed of a (6,8)-dihydroxyl- isocoumarin ring joined to tetrahydropyran group with a methyl moiety. (B) Inhibition of LlKRS, human-like LlKRS and SmKRS by cladosporin in enzyme assays. Percentage enzyme activity as a function of increasing inhibitor concentration (log scale, 0.01 nM—10 μM) is plotted using non-linear regression. These results represent the mean of three independent experiments performed in triplicates. (C) Protein thermal shift profile of LlKRS, SmKRS and human-like LlKRS (all three at 2 μM) in presence of cladosporin, or AMPPNP or without these two (but with L-lysine). The plot shows measured derivative Tm and data as plotted against fluorescence (arbitrary units) in y-axis and temperature in x-axis. Human-like LlKRS thermal shift at two concentrations of cladosporin 20 μM (in red) and 200 μM (in black) is shown. Mean data from triplicates are presented. (D) Binding data using ITC. Cladosporin was titrated into the protein samples and Kd was determined using Microcal origin software.
Table 1.
Isothermal titration calorimetry data showing strength of cladosporin binding.
Fig 3.
LlKRS-CLD-K complex structure and interactions.
(A) Dimer of LlKRS with bound cladosporin (CLD) and L-lysine (L-lys). Two monomers are denoted as molecules A and B. The anticodon binding domain (ABD), aminoacylation domain (AAD), CLD and L-lysine are depicted. (B) LlKRS monomer with bound cladosporin, L-lysine and motifs 1, 2 and 3 are highlighted in red. (C) View of experimental electron density at 1.2 σ (3.3 Å data) for cladosporin and L-lysine in LlKRS-K-CLD structure. (D) Cladosporin-interacting residues in binding pocket are shown. Phe344, Arg563 and His340 stack with the isocoumarin moiety of cladosporin. Hydroxyl groups from isocoumarin moiety form hydrogen bonds with Glu334 and Asn341. Methyl group joined to the THP ring points towards Ser346. (E) The bound L-lysine in active site is shown.
Table 2.
Data collection and refinement settings.
Fig 4.
Comparisons with HsKRS structure.
(A) Sequence alignment of LlKRS, SmKRS, HsKRS and PfKRS. Anticodon binding domain (ABD) and aminoacylation domain (AAD) sequences along with class II motifs 1, 2 and 3 are shown (in green). Eukaryotic insertions 1 and 2 are shown in red and purple boxes respectively. HsKRS tetramer interface 1 is highlighted in black and interface 2 is in blue. Cladosporin-selectivity residues Ser346 and Val329 are highlighted in brown. Disulfide-bonded cysteines (Cys517 and Cys540) in PfKRS and the orthologous residues in others are highlighted in yellow. (B) Cladosporin (CLD) and L-lysine (L-lys) bound HsKRS (4YCU) (in salmon) and LlKRS (green) PDBs are superimposed. Architectural differences in eukaryotic insertions and at HsKRS tetramer interface are shown.
Fig 5.
Cladosporin-binding mechanism of KRSs.
(A) Cladosporin binding in HsKRS (salmon) and LlKRS (green). Residues Ser346 and Val329 are replaced by larger Thr337 and Glu504 in HsKRS. Cladosporin bound to HsKRS structure is in blue and to LlKRS is in yellow. (B) Structural changes in apo-PfKRS (green) induced by cladosporin (CLD) and lysine (L-lys) individually are shown in blue and orange respectively. Cladosporin binding induces closing-in of the loop that contains motif 2, with rotameric adjustments in motif 2 residues Phe342, His338 and Arg559 (S1 Movie). This is accompanied by disulfide bond formation in the disordered loop (blue). L-lysine binding further induces a closing-in of mobile element present at roof of active site pocket and stabilization of the loop residues 580–590. The final PfKRS-CLD-K complex with all four major transitions is shown in orange. (C) HsKRS-CLD-K complex (cyan) overall conformation is similar to PfKRS-CLD-K complex (orange). The disulfide-stabilized loop is in an ordered helix in HsKRS. (D) LlKRS differs from previous KRS-cladosporin structures in that the incoming mobile roof and the disulfide regions are both disordered. Hence, cladosporin selectivity for P. falciparum, L. loa and perhaps S. mansoni KRSs is likely driven by the conserved residues Ser and Val that distally line active site pockets in these pathogen KRSs.
Fig 6.
Cladosporin derivatization strategy.
(A) Sequence alignment of schistosome and L. loa-related nematodes are shown. (B) Cartoon representation of the general reaction centers with substrates in KRS is shown. KRSs bind to ATP, L-lysine and CCA acceptor stem region of tRNA to carry aminoacylation reaction. Ap4A can be formed by KRSs as well. (C) Cladosporin derivatization to improve its ADME properties may focus on sites indicated with red arrows, or stereoisomeric alterations. (D) Cladosporin-based libraries may be useful across a spectrum of pathogens where KRS active sites and selectivity residues are conserved.