Structure of the Plasmodium falciparum PfA-M17_BES (3KR4)

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.

当前疟疾治疗与预防手段正面临严峻挑战，根源在于耐药疟原虫的快速出现。人类疟疾病原体恶性疟原虫（Plasmodium falciparum）表达两种中性氨肽酶（neutral aminopeptidases）：PfA-M1与PfA-M17，二者负责调控疟原虫在红细胞内寄生阶段生长发育所需的细胞内氨基酸池。这两种酶对疟原虫的生存至关重要，且已被验证为抗疟治疗的关键靶点。此前我们曾解析了单体PfA-M1的X射线晶体结构（X-ray crystal structure），并提出了底物进入活性位点以及游离氨基酸从该活性位点释放的作用机制。本研究报道了游离状态以及与两种具有抗疟活性的抑制剂结合状态下的六聚体亮氨酸氨肽酶（hexameric leucine aminopeptidase）PfA-M17的X射线晶体结构。PfA-M17六聚体的六个活性位点呈盘状排列，均向内朝向中央催化空腔；位于六个催化空腔入口处的柔性环（flexible loops）可调控底物的进入路径。与PfA-M1截然不同的是，PfA-M17拥有一个狭窄且疏水的一级特异性口袋，这正是其底物特异性高度受限的原因。我们还阐明了这两种酶的金属结合中心（metal-binding centres）在底物与药物结合过程中的核心作用：PfA-M17含有两个金属结合中心，而PfA-M1仅含一个。目前我们对PfA-M1与PfA-M17活性位点的深入解析，为开发新型双靶点及/或联合抗疟治疗方案提供了理性设计的可行路径。