Plasmodium falciparum Kelch13 and its artemisinin‐resistant mutants assemble as hexamers in solution: a SAXS data‐driven modelling study

Abstract

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.

ThePlasmodium falciparumartemisinin resistance-associated protein Kelch 13 is required for formation of normal cytostomes

“…Together, these data suggest that K13 is a component of the electron dense cytostomal collar and is essential for the formation or maintenance of this structure. The recent report that K13 forms a higher order oligomeric structure is also consistent with the possibility that K13 is a major repeating structural element in the cytostomal collar (Goel et al, 2022).…”

“…However, the most prevalent K13 mutations have no apparent effect on the ability of K13 to oligomerise (Goel et al, 2022). Other studies point to K13 mutations primarily impacting the parasite through modifying the overall abundance of K13 protein (Birnbaum et al, 2020;Siddiqui et al, 2017;Yang et al, 2019).…”

Saxs Data Based Glycosylated Models of Human Alpha-1-Acid Glycorprotein, a Key Player in Health, Disease and Drug Circulation

“…Programs used for data collection to processing are listed in Supplementary Table T1 . Direct desmearing was done using the beam profile and SAXSQuant program which provided SAXS dataset profiles representing pinhole camera profile 48 . Then, buffer subtraction was done from the solution SAXS profiles at different temperatures in auto mode.…”

“…This sample and matched buffer were used to acquire SAXS profiles from solution and buffer. All SAXS experiments were done at SAXSpace Instrument in CSIR-IMTECH equipped with sealed tube source in line collimation (Anton Paar Austria) [47][48][49] . Scattered X-rays were recorded on 1D Mythen detector (Dectris).…”

VISUALIZING THE NUCLEATING AND CAPPED STATES OF F-ACTIN BY Ca2+-GELSOLIN: SAXS DATA BASED STRUCTURES OF BINARY AND TERNARY COMPLEXES

“…Different models of the linker were computed using PEPFOLD3 server (https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/) [32]. Shape profiles of the 100 computed models were first checked visually for the curved shape seen earlier via SAXS based modelling, and then calculated SAXS profiles of the selected curved models were compared with SAXS profile of the linker extracted from dummy atom model as done for another case [33]. The best fit was ligated theoretically with the crystal structures of N- and C-halves of gelsolin.…”