Extended Data Fig. 9: Cryo-EM structure of the affinity-purified T. gondii complex III, obtained to elicit the binding mode of ELQ-300 at the Qi site reveals critical differences to quinolone-bound bovine structures.
From: Structure, assembly and inhibition of the Toxoplasma gondii respiratory chain supercomplex
(a) Schematic of the strategy used to C-terminally triple FLAG-epitope tag the Rieske protein. The expected size of integration PCRs are shown. (b) PCR to test integration of FLAG-epitope tag and CAT selection cassette into the endogenous locus, as outlined in (A). (c) Immunoblot analysis of whole cell lysate extracted from Rieske-3xFLAG and parental parasites. Samples were separated by SDS-PAGE, blotted, and detected using anti-FLAG and anti-MYS as a loading control. (d) Immunoblot analysis of fractions from FLAG immunoprecipitation. Equal cell equivalents of input, unbound (UB) and elute fractions were separated by SDS-PAGE and either stained with instant blue or blotted, and detected using anti-FLAG and anti-TOM40. (e) Purified complex III after one round of gel filtration: 5 µg of protein separated by SDS-PAGE and stained with instant blue. (f-j) Cryo-EM densities (transparent blue) for residues surrounding the Qi site showing the ELQ front view (F), back view (G), closeup on the cytochrome-b N-terminus (H) and conformers A and B of ELQ (I), closeup of a cardiolipin molecule (J). (k) In T. gondii (this study) the carbonyl and amino groups interact with H197 and D223, respectively. (l-m) In X-ray structures of the B. taurus homolog, the orientation of the 3-aryl quinolones is rotated by 180 degrees, leading to a different position of the diaryl-rest when compared to the parasite structure (L: PDB ID 5NMI, ref. 68; M: 6ZFS). Shown are inhibitors MJM170 (L) and WDH-1U-4 (M). (n-p) By comparison, owing to a flipped quinolone orientation 2-aryl-quinolones bind with the diaryl-like group facing in the same direction as in other structures of the bovine enzyme (N: PDB ID 7R3V; O: 5OKD; P: 6HAW), shown are inhibitors CK-2-67 (N), SCR0911 (O) and WDH2G7 (P). (q-r) Species-specific mutations in the apicomplexan Qi site explain susceptibility to ELQ-300. (q) ELQ-300 bound in the T. gondii cytochrome-b (this study, cryo-EM density in transparent blue) with overlaid a predicted model of the P. falciparum homolog (AlphaFold2). The apparent favorable interaction of the P. falciparum I22 with the methoxy group is indicated. (r) Van der Waals atomic radii of the 3-position chlorine and L26 shown in comparison with the smaller fluorine atom found in ELQ-316.
