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Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics.

Gupta A, Shah P, Haider A, Gupta K, Siddiqi MI, Ralph SA, Habib S - Open Biol (2014)

Bottom Line: We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion.A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms.Differences in predicted drug-ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.

ABSTRACT
Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug-ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.

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Modelling of antibiotic interactions with P. falciparum organelle ribosomes. (a) Azithromycin docked onto apicoplast (i) and mitochondrial (ii) ribosomes. As in the Thermus thermophilus ribosome–azithromycin structure, a single azithromycin molecule was docked at the binding site. (b) Interaction of clindamycin with apicoplast (i) and mitochondrial (ii) LSU rRNA. Bases that differ between the apicoplast and mitochondrial rRNA are shown in red and H-bonds as black lines. rRNA is in grey, L22 in cyan and antibiotics are in green.
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RSOB140045F3: Modelling of antibiotic interactions with P. falciparum organelle ribosomes. (a) Azithromycin docked onto apicoplast (i) and mitochondrial (ii) ribosomes. As in the Thermus thermophilus ribosome–azithromycin structure, a single azithromycin molecule was docked at the binding site. (b) Interaction of clindamycin with apicoplast (i) and mitochondrial (ii) LSU rRNA. Bases that differ between the apicoplast and mitochondrial rRNA are shown in red and H-bonds as black lines. rRNA is in grey, L22 in cyan and antibiotics are in green.

Mentions: Molecular docking of antibiotics was performed on P. falciparum apicoplast and mitochondrial ribosome models using Autodock4. Autodock uses grid-based energy evaluation for docking, where ligands are treated as flexible entities by exploring torsional degrees of freedom of ligand molecules. The first step of the Autodock algorithm involves conformational sampling of ligands followed by prediction and ranking of free energy of binding of these conformations. One hundred Autodock runs were performed for each inhibitor. To validate the reproducibility and sensitivity of the docking program, Autodock4 was used to dock the inhibitor co-complexed with the E. coli template. The inhibitor dock scores obtained for apicoplast and mitochondrial ribosomes are given in table 3. In the apicoplast, L22 located at the binding site for azithromycin [82] contains Arg88 that is predicted to form an H-bond with the inhibitor (figure 3a). Arg88 is replaced by Gly88 in mitochondrial L22 that does not form an H-bond with azithromycin. In addition, the rRNA sequence at the binding site also differs at two positions: A2612 and A2058 (E. coli number) in the apicoplast are replaced by C2612 and U2058, respectively, in the mitochondrion, a change that would alter the hydrophobic environment at the site. This might explain the differential specificity of azithromycin for organellar ribosomes. The higher affinity of the antibiotic for the apicoplast ribosome is also reflected in the lower dock scores obtained for azithromycin and the related macrolide erythromycin (table 3). Together with the LSU rRNA, L22 and L4 are predicted to form the peptide exit tunnel on the ribosome. The G76V mutation of apicoplast L4 has been reported to contribute to azithromycin resistance in P. falciparum lines [79] and modelling on the ribosome–azithromycin structure predicted a conformational shift in the side chain of Leu75 of L4 that could interfere with the azithromycin binding pocket. However, this model was constructed on the Deinococcus radiodurans (an extremophile bacterium) model that proposed the binding of two azithromycin residues at the site, one that interacted with the LSU rRNA and the other with L4, L22 and LSU rRNA [83]. Structures of the Haloarcula marismortui (an archaeon) and Thermus thermophilus large ribosomal subunits complexed with azithromycin have since led to the conclusion that a single molecule of the antibiotic binds to the ribosome [82]. This is supported by biochemical experiments that indicate that only one azithromycin molecule is bound to the E. coli ribosome [84]. No direct role for L4 in the interaction of azithromycin with P. falciparum apicoplast and mitochondrial ribosomes was detected in our model.Table 3.


Reduced ribosomes of the apicoplast and mitochondrion of Plasmodium spp. and predicted interactions with antibiotics.

Gupta A, Shah P, Haider A, Gupta K, Siddiqi MI, Ralph SA, Habib S - Open Biol (2014)

Modelling of antibiotic interactions with P. falciparum organelle ribosomes. (a) Azithromycin docked onto apicoplast (i) and mitochondrial (ii) ribosomes. As in the Thermus thermophilus ribosome–azithromycin structure, a single azithromycin molecule was docked at the binding site. (b) Interaction of clindamycin with apicoplast (i) and mitochondrial (ii) LSU rRNA. Bases that differ between the apicoplast and mitochondrial rRNA are shown in red and H-bonds as black lines. rRNA is in grey, L22 in cyan and antibiotics are in green.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4042851&req=5

RSOB140045F3: Modelling of antibiotic interactions with P. falciparum organelle ribosomes. (a) Azithromycin docked onto apicoplast (i) and mitochondrial (ii) ribosomes. As in the Thermus thermophilus ribosome–azithromycin structure, a single azithromycin molecule was docked at the binding site. (b) Interaction of clindamycin with apicoplast (i) and mitochondrial (ii) LSU rRNA. Bases that differ between the apicoplast and mitochondrial rRNA are shown in red and H-bonds as black lines. rRNA is in grey, L22 in cyan and antibiotics are in green.
Mentions: Molecular docking of antibiotics was performed on P. falciparum apicoplast and mitochondrial ribosome models using Autodock4. Autodock uses grid-based energy evaluation for docking, where ligands are treated as flexible entities by exploring torsional degrees of freedom of ligand molecules. The first step of the Autodock algorithm involves conformational sampling of ligands followed by prediction and ranking of free energy of binding of these conformations. One hundred Autodock runs were performed for each inhibitor. To validate the reproducibility and sensitivity of the docking program, Autodock4 was used to dock the inhibitor co-complexed with the E. coli template. The inhibitor dock scores obtained for apicoplast and mitochondrial ribosomes are given in table 3. In the apicoplast, L22 located at the binding site for azithromycin [82] contains Arg88 that is predicted to form an H-bond with the inhibitor (figure 3a). Arg88 is replaced by Gly88 in mitochondrial L22 that does not form an H-bond with azithromycin. In addition, the rRNA sequence at the binding site also differs at two positions: A2612 and A2058 (E. coli number) in the apicoplast are replaced by C2612 and U2058, respectively, in the mitochondrion, a change that would alter the hydrophobic environment at the site. This might explain the differential specificity of azithromycin for organellar ribosomes. The higher affinity of the antibiotic for the apicoplast ribosome is also reflected in the lower dock scores obtained for azithromycin and the related macrolide erythromycin (table 3). Together with the LSU rRNA, L22 and L4 are predicted to form the peptide exit tunnel on the ribosome. The G76V mutation of apicoplast L4 has been reported to contribute to azithromycin resistance in P. falciparum lines [79] and modelling on the ribosome–azithromycin structure predicted a conformational shift in the side chain of Leu75 of L4 that could interfere with the azithromycin binding pocket. However, this model was constructed on the Deinococcus radiodurans (an extremophile bacterium) model that proposed the binding of two azithromycin residues at the site, one that interacted with the LSU rRNA and the other with L4, L22 and LSU rRNA [83]. Structures of the Haloarcula marismortui (an archaeon) and Thermus thermophilus large ribosomal subunits complexed with azithromycin have since led to the conclusion that a single molecule of the antibiotic binds to the ribosome [82]. This is supported by biochemical experiments that indicate that only one azithromycin molecule is bound to the E. coli ribosome [84]. No direct role for L4 in the interaction of azithromycin with P. falciparum apicoplast and mitochondrial ribosomes was detected in our model.Table 3.

Bottom Line: We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion.A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms.Differences in predicted drug-ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular and Structural Biology, CSIR-Central Drug Research Institute, Lucknow, India.

ABSTRACT
Apicomplexan protists such as Plasmodium and Toxoplasma contain a mitochondrion and a relic plastid (apicoplast) that are sites of protein translation. Although there is emerging interest in the partitioning and function of translation factors that participate in apicoplast and mitochondrial peptide synthesis, the composition of organellar ribosomes remains to be elucidated. We carried out an analysis of the complement of core ribosomal protein subunits that are encoded by either the parasite organellar or nuclear genomes, accompanied by a survey of ribosome assembly factors for the apicoplast and mitochondrion. A cross-species comparison with other apicomplexan, algal and diatom species revealed compositional differences in apicomplexan organelle ribosomes and identified considerable reduction and divergence with ribosomes of bacteria or characterized organelle ribosomes from other organisms. We assembled structural models of sections of Plasmodium falciparum organellar ribosomes and predicted interactions with translation inhibitory antibiotics. Differences in predicted drug-ribosome interactions with some of the modelled structures suggested specificity of inhibition between the apicoplast and mitochondrion. Our results indicate that Plasmodium and Toxoplasma organellar ribosomes have a unique composition, resulting from the loss of several large and small subunit proteins accompanied by significant sequence and size divergences in parasite orthologues of ribosomal proteins.

Show MeSH