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

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.