Uneven spread of cis- and trans-editing aminoacyl-tRNA synthetase domains within translational compartments of P. falciparum

Abstract

Accuracy of aminoacylation is dependent on maintaining fidelity during attachment of amino acids to cognate tRNAs. Cis- and trans-editing protein factors impose quality control during protein translation, and 8 of 36 Plasmodium falciparum aminoacyl-tRNA synthetase (aaRS) assemblies contain canonical putative editing modules. Based on expression and localization profiles of these 8 aaRSs, we propose an asymmetric distribution between the parasite cytoplasm and its apicoplast of putative editing-domain containing aaRSs. We also show that the single copy alanyl- and threonyl-tRNA synthetases are dually targeted to parasite cytoplasm and apicoplast. This bipolar presence of two unique synthetases presents opportunity for inhibitor targeting their aminoacylation and editing activities in twin parasite compartments. We used this approach to identify specific inhibitors against the alanyl- and threonyl-tRNA synthetases. Further development of such inhibitors may lead to anti-parasitics which simultaneously block protein translation in two key parasite organelles, a strategy of wider applicability for pathogen control.

Figure 1. Distribution of putative editing domains in P. falciparum.

(a) Total number of P. falciparum putative editing-domain containing aaRSs, their amino acid specificities, their domain signatures and predicted localizations. Symbol * denotes the canonical P. falciparum aaRSs which lack discernible, typical editing domains. These were not investigated further in this work. (b) I, II, III, IV show active site architect of putative editing domains of Pf-Ed-LRS, Pf-Ed-IRS1, Pf-Ed-ARS and Pf-Ed-TRS showing the high level of conservation in critical residues involved in catalysis. PDB IDs are enclosed in brackets.

Figure 2. Targeting of P. falciparum aaRSs and of DTD.

Localization of (a) Pf-Ed-IRS1 (b) Pf-Ed-IRS2 (c) Pf-Ed-LRS (d) Pf-Ed-VRS (e) Pf-Ed-FRS (f) Pf-Ed-PRS (g) Pf-DTD. In all cases, upper panels show name of P. falciparum aminoacyl-tRNA synthetase, and their domain/ subdomain features. Middle panels show P. falciparum aminoacyl-tRNA synthetase expression in parasites (Par) and detection of recombinant P. falciparum aminoacyl- tRNA synthetase domains (Rec) by western blot analysis. Lower panels display their cellular localizations. Editing domains are colored yellow, aminoacylation domain (AA) is in red; RNA binding domain (RBD) is in green; ProRS specific C-terminal domain is in purple and un-annotated domains are in white. Blue arrow represents apicoplast targeting sequences predicted by PATS. Conserved motifs are highlighted by black strips. The parasite line used was GFP-tagged (strain D10 ACP_leader-GFP) where apicoplast fluorescence is in green. DAPI staining is in blue while aminoacyl-tRNA synthetases are stained with Alexa594 (red).

Figure 3. Expression and localization of Pf-Ed-TRS.

(a) Upper panel shows P. falciparum aminoacyl-tRNA synthetase name, and domain/ subdomain features. Middle panel shows P. falciparum aminoacyl-tRNA synthetase expression in parasites (Par) and detection of recombinant P. falciparum aminoacyl- tRNA synthetase domains (Rec) by western blot analysis. Lower panel displays their cellular localizations. Editing domains are colored yellow, aminoacylation domain (AA) is in red; RNA binding domain (RBD) is in green. Blue arrow represents apicoplast targeting sequences predicted by PATS. Conserved motifs are highlighted in black strips. (b) Upper and lower panels show confocal IFA with pre-immune sera and with anti-histidine antibodies, whereas the middle panels depict cytoplasmic staining of Pf-DTD. (c) Upper and lower panels show results of competitive confocal IFAs with rabbit anti-Ed-TRS antibodies which were pre-incubated with Ed-TRS in molar ratios of 1∶1 and 10∶1 respectively.

Figure 4. Expression, localization, modeling and inhibitor screening for Pf-Ed-ARS.

(a) Upper panels show P. falciparum aminoacyl-tRNA synthetase name, domain/ subdomain features. Middle panels show P. falciparum aminoacyl-tRNA synthetase expression in parasites (Par) and detection of recombinant P. falciparum aminoacyl- tRNA synthetase domains (Rec) by western blot analysis. Lower panels display their cellular localizations. The parasite line used was GFP-tagged (strain D10 ACP_leader-GFP) where apicoplast fluoresces green. DAPI staining is in blue while aminoacyl-tRNA synthetases are stained with Alexa594 (red). Editing domains are colored yellow, aminoacylation domain (AA) in red; RNA binding domain (RBD) is in green and the unidentified domain is colored in white. Blue arrow represents apicoplast targeting sequences predicted by PATS. Conserved motifs are highlighted in black strips. (b) Upper and lower panels show confocal IFA with pre-immune sera and with anti-histidine antibodies, whereas the middle panel depicts cytoplasmic staining of Pf-DTD. (c) Upper and lower panel show results of competitive IFAs with rabbit anti-Ed-ARS antibodies which were pre-incubated with Ed-ARS in molar ratios of 1∶1 and 10∶1 respectively. (d) Pf-Ed-ARS model with aminoacylation (AA) and editing domains (ED). The corresponding primary sequence domain structure is shown at bottom. AA motifs 1, 2 and 3 and ED motifs HXXXH and CXXXH are noted with red circles. (e) Pf-Ed-ARS-A5 inhibitor complex showing the docking of A5 within the aminoacylation domain of alanyl-tRNA synthetase. (f) Parasite growth inhibition assays using inhibitors (A1–A6, concentration range 1 nM to 1 mM) identified based on modeling of 3D structure. (g) IC₅₀ value calculation of A3 and A5 compounds by using two fold dilutions of the same compounds. (h–i) Cytotoxicity activity measurements using MTT assay in concentration range of 1 µM–200 µM.

Figure 5. Schematic illustration of cis- and trans- editing domains in P. falciparum.

The asymmetrical distribution of proofreading activities between parasite apicoplast and its cytoplasm can be gauged by the number of editing-domain containing aminoacyl- tRNA synthetases in each compartment. The single copy Pf-Ed-ARS and Pf-Ed-TRS were found to localize both to the cytoplasm and the apicoplast.