Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation

Rahman, A.; Tamseel, S.; Coppee, R.; Dutta, S.; Khan, N.; Faaiz, M.; Rastogi, H.; Nath, J. R.; Chowdhury, P.; Ashish, A.; Clain, J.; Bhattacharjee, S.

doi:10.1101/2024.01.23.576340

bioRxiv · 10.1101/2024.01.23.576340

Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation

Rahman, A.·Tamseel, S.·Coppee, R.·Dutta, S.·Khan, N.·Faaiz, M.·Rastogi, H.·Nath, J. R.·Chowdhury, P.·Ashish, A.·Clain, J.·Bhattacharjee, S.

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Abstract

The rapid emergence of artemisinin resistance (ART-R) poses a challenge to global malaria control efforts. ART potency is triggered by ferrous iron- and/or heme-mediated cleavage of the endoperoxide bond to generate reactive heme-ART alkoxy radicals and covalent heme-ART adducts that alkylate parasite targets or inhibit the detoxification of heme into {beta}-hematin crystals; both of which lead to parasite death. Mutations in the P. falciparum Kelch-containing protein Kelch13 (PfKekch13) confer clinical ART-R, in which the resistant parasites exhibit impaired hemoglobin uptake, reduced heme yield, and thus decreased ART activation. However, a more direct involvement of PfKelch13 in heme-mediated ART activation has not been reported. Here, we show that recombinant, purified PfKelch13 wild-type (WT) protein displays measurable binding affinity for both iron and heme, the main effectors for ART activation. Comparative biochemical analyses further indicate weaker heme-binding affinities in the two Southeast Asian ART-R PfKelch13 mutants C580Y and R539T compared to the ART-sensitive WT and A578S mutant proteins, which ultimately translates into reduced yield of heme-ART derivatives. In conclusion, this study provides the first evidence for regulated ART activation via the heme-binding propensity of PfKelch13, which may contribute towards modulating the level of ART-R in malaria parasites with PfKelch13 mutations.

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Rahman, A., Tamseel, S., Coppee, R., Dutta, S., Khan, N., Faaiz, M., Rastogi, H., Nath, J. R., Chowdhury, P., Ashish, A., Clain, J., Bhattacharjee, S.. 2024-01-24. Artemisinin-resistant Plasmodium falciparum Kelch13 mutant proteins display reduced heme-binding affinity and decreased artemisinin activation. https://doi.org/10.1101/2024.01.23.576340

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