Understanding the structural and functional requirements for substrate-induced conformational transitions of the Plasmodium falciparum chloroquine resistance transporter PfCRT

• Applicants: Professor Dr. Michael Lanzer; Professorin Dr. Rebecca Wade
• Subject Area: Parasitology and Biology of Tropical Infectious Disease Pathogens
• Term: since 2025
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 549643468

The Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) is a member of the drug/metabolite carrier family, sharing homology with mitochondrial thiol transporters found in the plant Arabidopsis thaliana. PfCRT holds a pivotal role in the physiology and pathophysiology of the human malaria parasite. It acts as a promiscuous oligopeptide carrier, facilitating access to essential nutrients crucial for parasite metabolic activities during intraerythrocytic development. Moreover, through the acquisition of multiple polymorphisms, PfCRT confers resistance to quinoline and quinoline-like antimalarial drugs by facilitating their efflux from their target organelle, the parasite digestive vacuole, thereby diminishing their efficacy. While recent years have seen advancements in our understanding of PfCRT's substrate specificity, the mechanistic details of the transport process remain elusive. Specifically, the mechanism by which PfCRT discriminates between substrates and non-substrates and facilitates their passage through its channel is poorly understood. Current consensus suggests PfCRT operates via an alternating access model, wherein substrate transport occurs in symport with protons through conformational switching between open-to-vacuole, occluded, and open-to-cytoplasm states. However, experimental evidence thus far only supports the open-to-vacuole conformation. The proposed study aims to elucidate the conformational dynamics underlying the transport cycle of PfCRT, delineate the routes taken by diverse substrates, and investigate changes in the protonation state of titratable residues throughout the transport process. Additionally, we seek to uncover how PfCRT coordinates proton translocation with ligand transport and assess the impact of PfCRT mutations on drug transport. To address these critical questions, we will employ a comprehensive approach, combining site-directed mutagenesis, functional expression of PfCRT variants in Xenopus laevis oocytes, transport assays with various substrates, and molecular dynamics simulations incorporating conventional and enhanced sampling techniques, supplemented by machine learning. Through elucidating these molecular mechanisms, we aim to deepen our understanding of PfCRT at both the molecular and mechanistic levels. By shedding light on the residues involved in substrate/drug binding and translocation, we anticipate contributing invaluable insights to the scientific community, thereby paving the way for innovative strategies in malaria treatment and drug development.

• DFG Programme: Research Grants