Department of Biochemistry, University of Calcutta, 35, Ballygunge Circular Road, Kolkata 700 019, West Bengal, India.
Mitra Tower, Lake Town, Block-A, Kolkata 700 089, India.
Infect Genet Evol. 2020 Nov;85:104418. doi: 10.1016/j.meegid.2020.104418. Epub 2020 Jun 16.
The pathological outcome of malaria due to Plasmodium falciparum infection depends largely on erythrocyte invasion by blood-stage merozoites which employ a cascade of interactions occurring between parasite ligands and RBC receptors. In a previous study exploring the genetic diversity of region-II of PfEBA-175, a ligand that plays a crucial part in parasite's RBC entry through Glycophorin A (GPA) receptor, we demonstrated that F2 domain of region-II underwent positive selection in Indian P. falciparum population through the accumulation of non-synonymous polymorphisms. Here, we examine the functional impact of two highly prevalent non-synonymous alterations in F2, namely Q584E & E592A, using a battery of molecular, biophysical and in-silico techniques. Application of circular dichroism, FTIR, fluorescence spectroscopy reveals that secondary and three-dimensional folding of recombinant-F2 protein carrying 584E and 592A residues (F2-Mut) differs significantly from that carrying 584Q and 592E (F2-3D7). A comparison of spectroscopic and thermodynamic parameters shows that F2-Mut is capable of forming a complex with GPA with higher efficiency compared to F2-3D7. In silico docking predicts both artemisinin and artesunate possess the capacity of slipping into the GPA binding crevices of PfEBA-175 and disrupt PfEBA-GPA association. However, the estimated affinity of artesunate towards PfEBA-175 with 584E and 592A residues is higher than that of artemisinin. Thermodynamic parameters computed using isotherms are concordant with this in-silico prediction. Together, our data suggest that the presence of amino acid alterations in F2 provide structural and functional stability favoring PfEBA-GPA interaction and artesunate can efficiently disrupt the interaction between GPA and PfEBA-175 even carrying altered amino acid residues. The present study alerts the malaria research community by presenting evidence that the parasite is gaining evolutionary fitness by cultivating genetic alterations in many of its proteins.
疟原虫感染导致的疟疾的病理结果在很大程度上取决于血期裂殖子对红细胞的入侵，裂殖子通过寄生虫配体和 RBC 受体之间发生的一系列相互作用来实现这一过程。在之前研究 PfEBA-175 区域-II 的遗传多样性的研究中，PfEBA-175 区域-II 是一种在寄生虫通过糖蛋白 A (GPA) 受体进入 RBC 过程中发挥关键作用的配体，我们证明该区域的 F2 结构域在印度恶性疟原虫种群中经历了正选择，这是通过积累非 synonymous多态性实现的。在这里，我们使用一系列分子、生物物理和计算机技术来研究 F2 中两个高度流行的非同义突变的功能影响。应用圆二色性、傅里叶变换红外光谱、荧光光谱学表明，携带 584E 和 592A 残基的重组-F2 蛋白（F2-Mut）的二级和三维折叠与携带 584Q 和 592E 残基的 F2-3D7 蛋白显著不同。比较光谱和热力学参数表明，F2-Mut 与 GPA 形成复合物的效率高于 F2-3D7。计算机对接预测青蒿素和青蒿琥酯都有能力滑入 PfEBA-175 的 GPA 结合缝隙并破坏 PfEBA-GPA 结合。然而，估算的青蒿琥酯与 584E 和 592A 残基的 PfEBA-175 的结合亲和力高于青蒿素。使用等温线计算的热力学参数与计算机预测结果一致。总的来说，我们的数据表明，F2 中的氨基酸改变提供了结构和功能稳定性，有利于 PfEBA-GPA 相互作用，青蒿琥酯可以有效地破坏 GPA 和 PfEBA-175 之间的相互作用，即使携带改变的氨基酸残基也是如此。本研究通过提供证据表明寄生虫通过在其许多蛋白质中培养遗传改变来获得进化适应性，从而引起疟疾研究界的关注。
