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Dryad

pH-dependence of the Plasmodium falciparum chloroquine resistance transporter is linked to the transport cycle

Data files

Jun 16, 2023 version files 23.08 MB

Abstract

This file contains the original data underpinning the following study:

The chloroquine resistance transporter, PfCRT, of the human malaria parasite Plasmodium falciparum displays a strong pH-sensitivity in the weakly acidic range. Consequently, PfCRT operates only at 60% of its maximal drug transport activity at the pH of 5.2 of the digestive vacuole, a proteolytic organelle from which PfCRT expels drugs that interfere with heme detoxification. Despite structural information, the molecular mechanism by which PfCRT senses pH changes has remained unclear. Here we show, by alanine-scanning mutagenesis and functional transport studies, that E207 plays a critical role in pH sensing. The E207A mutant displayed a pH-insensitive transport activity, while preserving drug substrate specificity. Replacement of E207 by Asp or His, but not by any other proteinogenic amino acid, reconstituted pH sensitivity. Molecular dynamics simulations and kinetics analyses suggest an allosteric binding model in which PfCRT can simultaneously accept both protons and chloroquine in a partial non-competitive manner, with increasing proton concentrations reducing the drug transport activity. Molecular dynamics simulations of the open-to-vacuole, the occluded and the open-to-cytosol conformation of PfCRT revealed a drastic relocation of E207 from a peripheral to an engaged location during the transport cycle, resulting in E207 forming a salt bridge with residue K80. We propose that the ionized carboxyl group of E207 acts as a hydrogen acceptor for interactions accelerating progression through the transport cycle and that the pH sensing is a by-product of this function.