The recently elucidated atomic resolution cryo-EM structure of the 7G8 isoform of Plasmodium falciparum chloroquine resistance transporter (PfCRT) suggests two pairs of proximal cysteine residues within the loop 7 (L7) domain. We wondered whether these might provide a redox active switch that might then regulate PfCRT function. Using site-specific mutagenesis, maleimide labeling, redox buffering, and chloroquine transport measurements, as well as molecular dynamics (MD) calculations, we probe the relative importance of all Dd2 PfCRT isoform C as well as their HS SH to S-S interconversion vs CQ transport function. Results show that CQ transport by PfCRT is regulated by the redox potential. We propose that disulfide bonds form at both the C289/C312 and C301/C309 pairs of Dd2 PfCRT and that these dynamic S-S bonds are required for full PfCRT CQ transport activity. Mutagenesis of all Dd2 PfCRT C to S or A reveals that no other C is functionally obligate but identifies C101, C139, C171, and C328 as involved in modulating CQ transport. Since two of the L7 C (C309 - C312) are within a CXXC motif (with X = D) that in theory can signify a metal binding site, we also model divalent metal ion binding using Metal3D and AlphaFold 3 and find that divalent metal may coordinate to C elsewhere in the protein but likely not to this CXXC motif. MD calculations done with 10 ns or 1 μs trajectories suggest large conformational changes in L7 near the initial drug binding site upon SH HS to S-S interconversion. Together, the data yield a model for how L7 disposed to the redox active digestive vacuole (DV) of the intraerythrocytic malarial parasite regulates PfCRT access to DV-disposed CQ2+.
Chernitsky-Hamd et al. (Wed,) studied this question.