Steered molecular dynamics simulations reveal critical residues for (un)binding of substrates, inhibitors and a product to the malarial M1 aminopeptidase

Malaria is a life-threatening disease spread by mosquitoes. Plasmodium falciparum M1 alanyl aminopeptidase (PfM1-AAP) is a promising target for the treatment of malaria. The recently solved crystal structures of PfM1-AAP revealed that the buried active site can be accessed through two channel openings: a short N-terminal channel with the length of 8 Å and a long C-terminal channel with the length of 30 Å. It is unclear, however, how substrates and inhibitors migrate to the active site and a product of cleavage leaves. Here, we study the molecular mechanism of substrate and inhibitor migration to the active site and the product release using steered molecular dynamics simulations. We identified a stepwise passage of substrates and inhibitors in the C-terminal channel of PfM1-AAP, involving (I) ligand recognition at the opening of the channel, (II) ionic translation to the ‘water reservoir’, (III) ligand reorientation in the ‘water reservoir’ and (IV) passage in a suitable conformation into the active site. Endorsed by enzymatic analysis of functional recombinant PfM1-AAP and mutagenesis studies, our novel ligand-residue binding network analysis has identified the functional residues controlling ligand migration within the C-terminal channel of PfM1-AAP. Furthermore, from unbinding simulations of the Arg product we propose a charge repulsion as the driving force to expel the product out from the N-terminal channel of PfM1-AAP. Our work paves the way towards the design of a novel class of PfM1-AAP inhibitors based on preventing substrate entry to the active site.

疟疾是一种经蚊子传播的致死性疾病。恶性疟原虫（Plasmodium falciparum）M1丙氨酰氨肽酶（PfM1-AAP）是抗疟疾治疗的极具潜力的药物靶点。近期解析的PfM1-AAP晶体结构显示，其包埋的活性位点可通过两个通道开口抵达：一条长度为8埃的短N端通道，以及一条长度为30埃的长C端通道。然而，目前尚不清楚底物与抑制剂如何迁移至活性位点，且裂解产物如何离开该活性位点的机制尚未阐明。本研究采用拉伸分子动力学（steered molecular dynamics）模拟方法，探究了底物与抑制剂向活性位点的迁移机制，以及产物的释放过程。我们发现，底物与抑制剂会通过PfM1-AAP的C端通道完成分步转运：(I) 配体在通道开口处被识别；(II) 经离子迁移抵达“水储库”；(III) 配体在“水储库”内发生构象重排；(IV) 以适宜构象进入活性位点。结合功能性重组PfM1-AAP的酶学分析与诱变实验结果，我们通过全新的配体-残基结合网络分析，鉴定出了调控PfM1-AAP C端通道内配体迁移的功能性残基。此外，通过精氨酸产物的解离模拟，我们提出电荷排斥作用是驱使产物从PfM1-AAP的N端通道排出的驱动力。本研究为基于阻断底物进入活性位点的新型PfM1-AAP抑制剂设计开辟了重要路径。