Structure of the Plasmodium falciparum PfA-M17 (3KQX)

Current therapeutics and prophylactics for malaria are under severe challenge as a result of the rapid emergence of drug-resistant parasites. The human malaria parasite Plasmodium falciparum expresses two neutral aminopeptidases, PfA-M1 and PfA-M17, which function in regulating the intracellular pool of amino acids required for growth and development inside the red blood cell. These enzymes are essential for parasite viability and are validated therapeutic targets. We previously reported the X-ray crystal structure of the monomeric PfA-M1 and proposed a mechanism for substrate entry and free amino acid release from the active site. Here we present the X-ray crystal structure of the hexameric leucine aminopeptidase, PfA-M17, alone and in complex with two inhibitors with anti-malarial activity. The six active sites of the PfA-M17 hexamer are arranged in a disc-like fashion so that they are orientated inwards to form a central catalytic cavity; flexible loops that sit at each of the six entrances to the catalytic cavern function to regulate substrate access. In stark contrast to PfA-M1, PfA-M17 has a narrow and hydrophobic primary specificity pocket which accounts for its highly restricted substrate specificity. We also explicate the essential roles for the metal-binding centres in these enzymes (two in PfA-M17 and one in PfA-M1) in both substrate and drug binding. Our detailed understanding of the PfA-M1 and PfA-M17 active sites now permits a rational approach in the development of a novel class of two-target and/or combination anti-malarial therapy.

当前针对疟疾的治疗和预防方法正面临着严峻的挑战，这主要归因于耐药寄生虫的快速出现。人类疟原虫恶性疟原虫表达两种中性氨基酸肽酶，即PfA-M1和PfA-M17，它们在调节红细胞内生长和发育所需的氨基酸库中发挥着作用。这些酶对于寄生虫的生存至关重要，并且已被确认为治疗靶点。我们先前报道了单聚体PfA-M1的X射线晶体结构，并提出了底物进入和自由氨基酸从活性位点释放的机制。在此，我们展示了六聚体亮氨酸氨基酸肽酶PfA-M17的X射线晶体结构，以及与两种具有抗疟活性的抑制剂复合的结构。PfA-M17六聚体的六个活性位点呈盘状排列，朝向内部形成中央催化腔；位于每个催化洞穴入口的柔性环状结构负责调节底物进入。与PfA-M1形成鲜明对比的是，PfA-M17具有狭窄且疏水的初级特异性口袋，这解释了其高度受限的底物特异性。我们还阐明了这些酶中金属结合中心（PfA-M17中两个，PfA-M1中一个）在底物和药物结合中的关键作用。我们对PfA-M1和PfA-M17活性位点的详细了解，现在允许我们采取理性方法开发一类针对两种靶点及/或联合抗疟疗法的创新疗法。