Structure-function analysis of plasmodium falciparum chloroquine resistance transporter in chloroquine resistance

Abstract

Malaria is a common and serious tropical disease spread by mosquitoes and affecting more than 100 countries in the world. There are approximately 250 million clinical cases and 1 million deaths due to malaria every year, especially amongst children and pregnant women. Chloroquine (CQ) has been an effective drug against malaria but its intensive use has led to the emergence of chloroquine resistance (CQR) since 1970s', resulting in a dramatic increase in mortality and morbidity of malaria. Recent studies have identified a gene, Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene, as the determinant gene for CQR. The exact mechanism of CQR mediated by PfCRT is not yet clear, but it is believed that the loss of positive charge due to K76T point mutation at CQR PfCRT allows CQ to escape from the parasite digestive vacuole (DV). The natural functions and substrates of PfCRT are unknown but it has been proposed that PfCRT plays an important role in CQ transport and intracellular pH (pHi) regulation in parasite. In order to study the functions of PfCRT, mutants of PfCRT were generated, expressed in Pichia pastoris (P. pastoris), purified and reconstituted into proteoliposomes (PLs) for various functional studies. PfCRT mutants were able to accumulate radiolabeled [3H]-CQ into microsomes, in an ATP-dependent, verapamil (VP)-and nigericin-inhibitable manner, which was consistent with the CQR phoneotypes observed in P. falciparum. PfCRT PLs retained the CQ transport activities of microsomes, though the activity was 5 times lower, suggesting that full activity of CQ transport could not be explained by PfCRT alone. However, full level of CQ transport activities of PfCRT PLs could be restored when reconstituted in the presence of other yeast proteins, suggesting that there are other yeast proteins that might assist the folding and/or functioning of PfCRT. PfCRT mutants also showed an ability to regulate pHi in response to CQ and ATP/ADP/AMP, which was not observed in PfCRT PLs. It indicated that the pHi regulatory effects observed were not due to PfCRT alone, but also the presence of yeast proteins. Using a photoreactive, radiolabeled ATP analogue, [α32P]8N3ATP, it was shown that PfCRT was not an ATP-binding protein. However, it seemed that PfCRT was able to interact with a yeast protein of ~50kDa at ATP-binding domain, blocking the [α32P]8N3ATP-crosslinking at that particular protein. The yeast proteins that physically interacted with PfCRT were later identified as α-and β-subunit of F1Fo-ATP synthase by mass spectrometry and the interaction was confirmed by immunoprecipitation (IP). The interaction between PfCRT, α-and β-subunit of F1Fo-ATP synthase might explain the ATP-dependent CQ transport by PfCRT and might also indicate a novel function of PfCRT in regulating pHi. Further investigation on protein interaction of PfCRT in parasite using same approach may help understanding the mechanism of PfCRT’s function and CQR.