Reconstructing the Qo site of Plasmodium falciparum bc 1 complex in the yeast enzyme

Abstract

The bc 1 complex of the mitochondrial respiratory chain is essential for Plasmodium falciparum proliferation, the causative agent of human malaria. Therefore, this enzyme is an attractive target for antimalarials. However, biochemical investigations of the parasite enzyme needed for the study of new drugs are challenging. In order to facilitate the study of new compounds targeting the enzyme, we are modifying the inhibitor binding sites of the yeast Saccharomyces cerevisiae to generate a complex that mimics the P. falciparum enzyme. In this study we focused on its Qo pocket, the site of atovaquone binding which is a leading antimalarial drug used in treatment and causal prophylaxis. We constructed and studied a series of mutants with modified Qo sites where yeast residues have been replaced by P. falciparum equivalents, or, for comparison, by human equivalents. Mitochondria were prepared from the yeast Plasmodium-like and human-like Qo mutants. We measured the bc 1 complex sensitivity to atovaquone, azoxystrobin, a Qo site targeting fungicide active against P. falciparum and RCQ06, a quinolone-derivative inhibitor of P. falciparum bc 1 complex.The data obtained highlighted variations in the Qo site that could explain the differences in inhibitor sensitivity between yeast, plasmodial and human enzymes. We showed that the yeast Plasmodium-like Qo mutants could be useful and easy-to-use tools for the study of that class of antimalarials.

Figure 1. Sequence and structure of the Q_o site.

(A) Comparison of cytochrome b sequence. Regions of the polypeptide forming the Q_o domain and its vicinity are shown. The Q_o site itself is formed by residues located in the regions 120–150 and 260–280. Mutated residues studied here are highlighted in colour. Green arrows and bars indicate structural components, as shown in panels B and C: C and F1 are transmembrane helices; cd1 and cd2, extramembrane short helices; ef, a loop containing highly conserved residues. Hs, human; Sc, yeast; Pf, Plasmodium falciparum. (B) Location of the mutated residues in the yeast bc ₁ Q_o site. The figure was drawn using the coordinates of 3CX5.PDB . VLPW, location of the mutation CCVY_133–136VLPW. (C) Molecular model of atovaquone (lilac CPK) and RCQ06 (cyan CPK) docked in the Q_o site of yeast cytochrome b (3CX5.PDB [39]). Selected sidechains from cytochrome b and the ISP are represented in orange. The alpha carbon backbones of cytochrome b and the ISP are represented in cartoon form in grey/green and dark pink respectively. Helices of interest within cytochrome b forming structural elements of Q_o are labelled in green. Also shown are the haem b _l and [2Fe2S] prosthetic groups of cytochrome b and the ISP. Residue notation corresponds to the yeast enzyme. Atv, atovaquone.

Figure 2. Chemical structures of atovaquone, azoxystrobin and RCQ06.