Malaria is a mosquito borne infectious disease caused by a unicellular Apicomplexan parasite of the Plasmodia genus. The emergence and subsequent spread of drug resistance in the highly virulent Plasmodium falciparum parasite has been a major setback in eradicating malaria, which affects an estimated 216 million individuals and causes 445,000 deaths annually worldwide. Chloroquine (CQ) was once used as the first-line antimalarial drug treatment, until CQ-resistant parasites emerged in endemic regions including Africa, Southeast Asia, and South America. More recently, parasites have developed resistance to the current first line drug piperaquine (PPQ), used in combination with dihydroartemisinin (DHA) in Southeast Asia.
Plasmodium falciparum chloroquine resistance transporter (PfCRT), a member of the drug/metabolite transporter (DMT) superfamily, is a 49-kDa integral transmembrane protein localized in the digestive vacuole (DV) of the pathogenic parasite. Mutations in PfCRT have been identified as the core determinants of Plasmodium falciparum resistance to CQ and PPQ by mediating the efflux of these antimalarial drugs. All CQ resistance-conferring PfCRT isoforms share the K76T mutation, which is widely used as a molecular marker for CQ resistance. Despite the significance in the impact of drug-resistant malaria, a detailed understanding of PfCRT physiological function and the molecular basis of PfCRT-mediated drug resistance have been hampered by a lack of high-resolution structural information. This dissertation describes the first structure of PfCRT and reveals the interaction of drugs with the purified and reconstituted protein.
We determined the structure of the 49-kDa PfCRT 7G8, a clinically relevant CQ-resistant isoform found in South America, to 3.2 Å resolution by single-particle cryo-electron microscopy (cryo-EM), in complex with a specific antigen-binding fragment (Fab) to overcome current size limitations in cryo-EM. Our PfCRT structure displays an inward-open conformation, consists of 10 transmembrane (TM) helices with an inverted topology, and has unique elements including two juxtamembrane helices and a highly conserved cysteine-rich loop between TM helix 7 and 8. The architecture of PfCRT is similar to other members of the DMT superfamily. TM helices 1-4 and 6-9 in PfCRT form a central cavity which is a potential binding site for both CQ and PPQ. A striking feature is that virtually all the CQ resistance mutations, identified from decades of investigation into PfCRT variants that have evolved independently across the malaria-endemic world, map around this central, negatively-charged cavity. Distinct mutations that have been proposed to cause high-level PPQ resistance in parasites, which cause a loss of CQ resistance, form a planar ring that also lines this cavity. Functional experiments with various purified PfCRT isoforms or mutants provide evidence that drug resistance is possibly due to pH- and membrane potential-dependent drug transport. We also show that PfCRT CQ-resistant isoforms bind and transport arginine, suggesting that positively charged amino acids may be putative transport substrates for CQ-resistant PfCRT. This work provides a structural and functional framework to understand the mechanism of PfCRT-mediated drug resistance in the malaria parasite.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-c5s0-ff42 |
| Date | January 2019 |
| Creators | Kim, Jonathan Young |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
Page generated in 0.0025 seconds