Abstract
Numerous mutations in the Plasmodium falciparum Kelch13 (K13) protein confer resistance to artemisinin derivatives, the current front-line antimalarial drugs. K13 is an essential protein that contains BTB and Kelch-repeat propeller (KREP) domains usually found in E3 ubiquitin ligase complexes that target substrate protein(s) for ubiquitin-dependent degradation. K13 is thought to bind substrate proteins, but its functional/interaction sites and the structural alterations associated with artemisinin resistance mutations remain unknown. Here, we screened for the most evolutionarily conserved sites in the protein structure of K13 as indicators of structural and/or functional constraints. We inferred structure-dependent substitution rates at each amino acid site of the highly conserved K13 protein during the evolution of Apicomplexa parasites. We found two solvent-exposed patches of extraordinarily conserved sites likely involved in protein-protein interactions, one in BTB and the other one in KREP. The conserved patch in K13 KREP overlaps with a shallow pocket that displays a differential electrostatic surface potential, relative to neighboring sites, and that is rich in serine and arginine residues. Comparative structural and evolutionary analyses revealed that these properties were also found in the functionally-validated shallow pocket of other KREPs including that of the cancer-related KEAP1 protein. Finally, molecular dynamics simulations carried out on PfK13 R539T and C580Y artemisinin resistance mutant structures revealed some local structural destabilization of KREP but not in its shallow pocket. These findings open new avenues of research on one of the most enigmatic malaria proteins with the utmost clinical importance.
Highlights
Current efforts to control malaria are threatened by the spread in Southeast Asia (SEA) of Plasmodium falciparum parasites that are resistant to artemisinin derivatives (ARTs)[1]
People usually search for amino acid sites that have evolved more rapidly than expected under a neutral model and interpret them as a signature of adaptive evolution corresponding to a gain of new function(s)[46,47]
In the context of a sustained and intense purifying selection operating in all annotated domains of K13, our inter-species analysis of K13 sequence evolution coupled to the BTB-Kelch-repeat propeller (KREP) tertiary structure of PfK13 identified two patches of slowly evolving sites
Summary
Current efforts to control malaria are threatened by the spread in Southeast Asia (SEA) of Plasmodium falciparum parasites that are resistant to artemisinin derivatives (ARTs)[1]. Naturally-occurring pfk[13] mutants associate with several key features: an increased expression of unfolded protein response pathways, detected by the in vivo transcriptomics analysis of 1,043 P. falciparum isolates from patients with acute malaria response[16]; lower levels of ubiquitinated proteins17,18; 2-fold lower abundance of PfK13 protein, as measured by quantitative dimethyl-based proteomics analysis of Cambodian isogenic strains[19] (note that a different result was reported for two African isogenic strains20); and a basal, constitutive phosphorylation of the parasite eukaryotic initiation factor-2α (eIF2α) at the early intraerythrocytic stage, which controls the repression of general translation and ART-induced latency[15,21]. The increased abundance of PfPI3K, and its lipid product PfPI3P, in mutant parasites may influence host cell remodeling and neutralize ART toxic proteopathy[17,20] These experimental data and the Gene Ontology annotation of PfK13, which contains BTB and KREP domains, are consistent with the hypothesis that PfK13 may function as an E3 ubiquitin ligase substrate adaptor (Fig. 1b)[17,21,22,23]. PfK13 may bind several substrates, in addition to PfPI3K
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