Structures of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) Isoforms and Their Interactions with Chloroquine

Abstract

Using a recently elucidated atomic-resolution cryogenic electron microscopy (cryo-EM) structure for the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein 7G8 isoform as template et al. Nature 2019, 576, 315−320], we use Monte Carlo molecular dynamics (MC/MD) simulations of PfCRT embedded in a 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to solve energy-minimized structures for 7G8 PfCRT and two additional PfCRT isoforms that harbor 5 or 7 amino acid substitutions relative to 7G8 PfCRT. Guided by drug binding previously defined using chloroquine (CQ) photoaffinity probe labeling, we also use MC/MD energy minimization to elucidate likely CQ binding geometries for the three membrane-embedded isoforms. We inventory salt bridges and hydrogen bonds in these structures and summarize how the limited changes in primary sequence subtly perturb local PfCRT isoform structure. In addition, we use the "AlphaFold" artificial intelligence AlphaFold2 (AF2) algorithm to solve for domain structure that was not resolved in the previously reported 7G8 PfCRT cryo-EM structure, and perform MC/MD energy minimization for the membrane-embedded AF2 structures of all three PfCRT isoforms. We compare energy-minimized structures generated using cryo-EM vs AF2 templates. The results suggest how amino acid substitutions in drug resistance-associated isoforms of PfCRT influence PfCRT structure and CQ transport.

PfCRT mutations conferring piperaquine resistance in falciparum malaria shape the kinetics of quinoline drug binding and transport

“…Multiple sequence alignments of different Plasmodium CRT homologs and orthologues show a high degree of conservation in the vacuolar loop, suggesting a mechanistic role [ 9 ]. The area of the transporter facing the digestive vacuole, near the vacuolar loops, was proposed as the initial binding site for CQ [ 42 ], thus supporting a possible role of the loop in the initial phase of substrate binding. Several membrane transporters are known to have extracellular loops acting as gates and taking part in the whole transport mechanisms [ 43 ], thus supporting the idea that such behavior could hold true for PfCRT.…”

Additional PfCRT mutations driven by selective pressure for improved fitness can result in the loss of piperaquine resistance and altered Plasmodium falciparum physiology

“…Subsequently, we used Monte Carlo Energy Minimization Molecular Dynamics to predict how the individual amino acid substitutions found in the three novel PfCRT isoforms might perturb local protein structure and drug interactions ( 32 ). As expected, the structures of the four PfCRT isoforms studied herein (Dd2 + F145I, Dd2 + F145I + F131C, Dd2 + F145I + C258W, and Dd2 + F145I + I347T) shared many similarities with the CQ-resistant Dd2 isoform from which they were derived ( Fig.…”

“…Nonetheless, the minor local differences between isoforms provide insight into their different PPQ binding or transport properties (PPQ is similar in structure to a CQ “dimer”), relative to CQ ( 18 ). To identify local differences in structure that may be relevant for the altered parasite phenotypes mediated by these novel PfCRT isoforms, we performed molecular dynamics analyses, as earlier applied to PfCRT to inventory residue side chain-side chain hydrogen bonds (HB) and salt bridges (SB) ( 32 ). Key differences between the isoforms are summarized in Table 2 with a full inventory of SB provided in Table S4.…”

APlasmodium falciparumgenetic cross reveals the contributions ofpfcrtandplasmepsin II/IIIto piperaquine drug resistance

“…Reported drug resistance-associated mutations in PfCRT generally reside in one of the 10 transmembrane (TM) domains and, specifically, in regions that make up the central cavity of the transporter (33, 46). The G367C substitution is distinct from experimentally validated PPQ- R-associated mutations in that it resides in the digestive vacuole (DV)-exposed portion of TM9 and is not part of the core structure of the transporter’s central cavity (27, 28, 33, 47, 48) (Figure S3).…”

“…This parasite carries a Dd2-like pfcrt with the addition of a novel mutation G367C. Most drug resistance-associated mutations in PfCRT are found in one of the 10 transmembrane (TM) domains, and specifically regions that make up the central cavity of the transporter (33,38). The G36C substitution differs from other PPQ-R-associated mutations in that while it is found within TM9 with other known mutations, it is found in the digestive vacuole (DV) exposed portion of this protein and does not part of the core structure of the transporter's central cavity (Supplementary Figure 2).…”