Université de Paris, UMR 261 MERIT, IRD, F-75006 Paris, France.
Department of Biology and York Biomedical Research Institute, University of York, Wentworth Way, York, YO10 5DD, UK.
Sci Rep. 2019 Jul 23;9(1):10675. doi: 10.1038/s41598-019-47034-6.
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.
疟原虫 falciparum Kelch13（K13）蛋白的许多突变赋予了对青蒿素衍生物的抗性，青蒿素衍生物是目前的一线抗疟药物。K13 是一种必需蛋白，含有 BTB 和 Kelch 重复推进器（KREP）结构域，通常存在于 E3 泛素连接酶复合物中，该复合物将靶标蛋白（s）用于泛素依赖性降解。K13 被认为与底物蛋白结合，但它的功能/相互作用位点以及与青蒿素抗性突变相关的结构改变仍然未知。在这里，我们筛选了 K13 蛋白结构中最进化保守的位点，作为结构和/或功能约束的指标。我们推断了在 Apicomplexa 寄生虫的进化过程中，高度保守的 K13 蛋白中每个氨基酸位点的结构依赖性取代率。我们发现了两个暴露在溶剂中的特别保守的位点，可能参与了蛋白质-蛋白质相互作用，一个在 BTB 上，另一个在 KREP 上。K13 KREP 中的保守斑块与一个浅口袋重叠，该口袋显示出与相邻位点相比的差异静电表面电势，并且富含丝氨酸和精氨酸残基。比较结构和进化分析表明，这些特性也存在于其他 KREP 中的功能验证的浅口袋中，包括癌症相关的 KEAP1 蛋白。最后，对 PfK13 R539T 和 C580Y 青蒿素抗性突变体结构进行的分子动力学模拟显示，KREP 的一些局部结构不稳定，但在其浅口袋中没有。这些发现为最神秘的疟疾蛋白之一开辟了新的研究途径，该蛋白具有最大的临床重要性。
