The artemisinin‐resistant mutations in Plasmodium falciparum (PfKelch13) identified worldwide are mostly confined to the Broad‐complex, tramtrack and bric‐à‐brac/poxvirus and zinc‐finger (BTB/POZ) and Kelch‐repeat propeller (KRP) domains. To date, only two crystal structures of the BTB/POZ‐KRP domains as tight dimers are available, which limits structure‐based predictions and interpretation of its role(s) in inducing clinical artemisinin resistance. Our solution Small‐Angle X‐ray Scattering (SAXS) data analysis and shape restoration brought forth that: (a) PfKelch13 forms a stable hexamer in P6 symmetry, (b) interactions of the N‐termini drive the hexameric assembly, and (c) the six KRP domains project independently in space, forming a cauldron‐like architecture. We further deduce that the artemisinin‐sensitive mutant A578S is packed like the wild‐type protein, however, hexameric assemblies of the predominant artemisinin‐resistant mutants R539T and C580Y displayed detectable differences in the spatial positioning of their BTB/POZ‐KRP domains. Lastly, mapping of mutations known to enable artemisinin resistance suggested evolutionary pressure in the selection for mutations in the BTB/POZ‐KRP domains. These mutations appear non‐detrimental to the hexameric assembly of proteins, and yet somehow alter the flux of downstream events essential for the susceptibility to artemisinin.
Artemisinin-resistant mutations in PfKelch13 identified worldwide are mostly confined to its BTB/POZ and KRP domains. To date, only two crystal structures of the BTB/POZ-KRP domains as tight dimers are available, which limits structure-based interpretations of its functionality. Our solution Small-Angle X-ray Scattering (SAXS) data driven shape restoration of larger length of protein brought forth that: i) PfKelch13 forms a stable hexamer in P6 symmetry, ii) interactions of the N-termini drive the hexameric assembly, and iii) the six KRP domains project independently in space, forming a cauldron-like architecture. While artemisinin-sensitive mutant A578S packed like the wild-type, hexameric assemblies of dominant artemisinin-resistant mutant proteins R539T and C580Y displayed detectable differences in spatial positioning of their BTB/POZ-KRP domains. Lastly, mapping of mutations known to enable artemisinin resistance explained that most mutations exist mainly in these domains because they are non-detrimental to assembly of mutant PfKelch13 and yet can alter the flux of downstream events essential for susceptibility to artemisinin.
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