Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) Is a Redox-Dependent Drug Transporter

High Resolution Image Download MS PowerPoint Slide 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 CQ 2+ .