Cryo-EM structure of the Plasmodium falciparum 80S ribosome bound to the anti-protozoan drug emetine

Abstract

Malaria inflicts an enormous burden on global human health. The emergence of parasite resistance to front-line drugs has prompted a renewed focus on the repositioning of clinically approved drugs as potential anti-malarial therapies. Antibiotics that inhibit protein translation are promising candidates for repositioning. We have solved the cryo-EM structure of the cytoplasmic ribosome from the human malaria parasite, Plasmodium falciparum, in complex with emetine at 3.2 Å resolution. Emetine is an anti-protozoan drug used in the treatment of ameobiasis that also displays potent anti-malarial activity. Emetine interacts with the E-site of the ribosomal small subunit and shares a similar binding site with the antibiotic pactamycin, thereby delivering its therapeutic effect by blocking mRNA/tRNA translocation. As the first cryo-EM structure that visualizes an antibiotic bound to any ribosome at atomic resolution, this establishes cryo-EM as a powerful tool for screening and guiding the design of drugs that target parasite translation machinery.

Keywords: Plasmodium falciparum; biophysics; cryo-EM; drug development; malaria; ribosome; structural biology.

Figure 1.. Cryo-EM data and processing.

(A) Sucrose gradient purification of Pf80S ribosomes. (B) Representative electron micrograph showing Pf80S particles. (C) Fourier Shell Correlation (FSC) curves indicating the overall resolutions of unmasked (red), Pf40S masked (green) and Pf60S masked (blue) reconstructions of the Pf80S–emetine complex. (D) Representative density with built models of a β-strand with well-resolved side chains (left), an RNA segment with separated bases (middle), and a magnesium ion (green sphere) that is coordinated by RNA backbone phosphates. (E) Density maps colored according to local resolution for the unmasked Pf80S (left) and masked Pf40S and Pf60S subunits (right). DOI: http://dx.doi.org/10.7554/eLife.03080.003

Figure 1—figure supplement 1.. FSC curves between the final refined atomic model and the reconstructions from all particles (black); between the model refined in the reconstruction from only half of the particles and the reconstruction from that same half (FSC_work, red); and between that same model and the reconstruction from the other half of the particles (FSC_test, green), for Pf40S (A) and Pf60S (B).

DOI: http://dx.doi.org/10.7554/eLife.03080.004

Figure 2.. Structure of the Pf80S ribosome.

Overview of Pf80S atomic model showing views facing (A) tRNA entry side and (B) tRNA exit side. rRNAs are shown in gray, proteins numbered according to Ban et al. (2014). (C and D) Pf40S and Pf60S subunits are colored in yellow and blue respectively. Flexible regions are shown in red and at a resolution of 6 Å. Pf-specific expansion segments (ESs) relative to human ribosomes are labeled. Their numbering is as described for the human cytoplasmic ribosome (Anger et al., 2013). DOI: http://dx.doi.org/10.7554/eLife.03080.005

Figure 2—figure supplement 1.. Secondary structure of Pf18S rRNAs.

Pf-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (Cannone et al., 2002). DOI: http://dx.doi.org/10.7554/eLife.03080.006

Figure 2—figure supplement 2.. Secondary structure of the 5′ half of Pf 28S rRNA.

Pf-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (Cannone et al., 2002). DOI: http://dx.doi.org/10.7554/eLife.03080.007

Figure 2—figure supplement 3.. Secondary structure of the 3′ half of Pf28S rRNA.

Pf-specific ESs are highlighted in a labeled red box. Regions not built in the atomic model are colored in blue text. The secondary structure was modified from the CRW site (Cannone et al., 2002). DOI: http://dx.doi.org/10.7554/eLife.03080.008

Figure 3.. Details of Pf-specific protein extensions and rRNA ESs near the (A and B) subunit interface (C) P stalk and (D) the L1 stalk.

Pf-specific elements are shown in red. DOI: http://dx.doi.org/10.7554/eLife.03080.013

Figure 4.. Emetine binds to the E-site of the Pf40S subunit.

(A) 2D chemical structure of emetine. (B) A 4.5 Å filtered difference map (red density) at 5 standard deviation overlaid with the Pf80S map filtered at 6 Å (blue and yellow for Pf60S and Pf40S respectively), showing the emetine density at the E-site of the Pf40S. The emetine binding site in (C) empty and (D) emetine-bound structures, with (E) density for emetine alone at 3.2 Å. DOI: http://dx.doi.org/10.7554/eLife.03080.014

Figure 5.. Molecular details of the emetine–ribosome interaction.

(A) Overview of emetine at the binding interface formed by the three conserved rRNA helices and uS11. h23 is in green, h24 in cyan, h45 in blue, uS11 in pink, and emetine in yellow. (B) 2D representation showing the interaction of emetine with binding residues. Substitution contour represents potential space for chemical modification of emetine. (C) Residues in physical contact with emetine. Hydrogen bond is indicated as dashes. DOI: http://dx.doi.org/10.7554/eLife.03080.015

Figure 5—figure supplement 1.. Comparison of the emetine binding residues between Pf80S and human ribosomes.

Human and Pf-specific elements are colored in yellow and cyan respectively, with Pf numbering. Emetine is in purple. DOI: http://dx.doi.org/10.7554/eLife.03080.016

Figure 6.. Comparison with pactamycin.

Superposition of emetine and pactamycin at the Pf40S emetine binding pocket. Emetine and pactamycin are shown in yellow and red respectively. DOI: http://dx.doi.org/10.7554/eLife.03080.017