The FK506-binding protein of Plasmodium knowlesi (Pk-FKBP35) is considerably a viable antimalarial drug target, which belongs to the peptidyl-prolyl cis-trans isomerase (PPIase) protein family member. Structurally, this protein consists of an N-terminal FK506-binding domain (FKBD) and a C-terminal tetratricopeptide repeat domain (TPRD). This study aims to decipher functional properties of these domains as a platform for development of novel antimalarial drugs. Accordingly, full-length Pk-FKBP35 as well as its isolated domains, Pk-FKBD and Pk-TPRD were overexpressed, purified, and characterized. The results showed that catalytic PPIase activity was confined to the full-length Pk-FKBP35 and Pk-FKBD, suggesting that the catalytic activity is structurally regulated by the FKBD. Meanwhile, oligomerization analysis revealed that Pk-TPRD is essential for dimerization. Asp55, Arg60, Trp77 and Phe117 in the Pk-FKBD were considerably important for catalysis as underlined by significant reduction of PPIase activity upon mutations at these residues. Further, inhibition activity of Pk-FKBP35 towards calcineurin phosphatase activity revealed that the presence of FKBD is essential for the inhibitory property, while TPRD may be important for efficient binding to calcineurin. We then discussed possible roles of FKBP35 in Plasmodium cells and proposed mechanisms by which the immunosuppressive drug, FK506, interacts with the protein.

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诺氏疟原虫FK506结合蛋白35的功能域的表达和表征

诺氏疟原虫的FK506结合蛋白（Pk-FKBP35）是相当可行的抗疟药物靶标，属于肽基-脯氨酰顺反异构酶（PPIase）蛋白家族成员。在结构上，该蛋白质由N端FK506结合结构域（FKBD）和C端四肽重复结构域（TPRD）组成。这项研究旨在破译这些域的功能特性，作为开发新型抗疟疾药物的平台。因此，全长Pk-FKBP35及其分离的结构域Pk-FKBD和Pk-TPRD被过表达，纯化和鉴定。结果表明，催化PPIase活性仅限于全长Pk-FKBP35和Pk-FKBD，表明催化活性受FKBD结构调节。同时，寡聚分析表明，Pk-TPRD对于二聚作用至关重要。Pk-FKBD中的Asp55，Arg60，Trp77和Phe117对于催化非常重要，因为这些残基突变后PPIase活性显着降低，从而突显了这一点。此外，Pk-FKBP35对钙调磷酸酶磷酸酶活性的抑制活性表明，FKBD的存在对于抑制特性至关重要，而TPRD对于有效结合钙调磷酸酶可能很重要。然后，我们讨论了FKBP35在疟原虫细胞中的可能作用，并提出了免疫抑制药物FK506与蛋白质相互作用的机制。Pk-FKBP35对钙调磷酸酶磷酸酶活性的抑制活性表明，FKBD的存在对于抑制特性至关重要，而TPRD对于有效结合钙调磷酸酶可能很重要。然后，我们讨论了FKBP35在疟原虫细胞中的可能作用，并提出了免疫抑制药物FK506与蛋白质相互作用的机制。Pk-FKBP35对钙调磷酸酶磷酸酶活性的抑制活性表明，FKBD的存在对于抑制特性至关重要，而TPRD对于有效结合钙调磷酸酶可能是重要的。然后，我们讨论了FKBP35在疟原虫细胞中的可能作用，并提出了免疫抑制药物FK506与蛋白质相互作用的机制。