Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson’s disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

中文翻译：

---

一种针对帕金森病相关 LRRK2 的设计锚蛋白重复蛋白


富含亮氨酸重复激酶 2 (LRRK2) 是一种大型多结构域蛋白，包含两个催化结构域（一个激酶和一个 GTP 酶）以及蛋白质相互作用结构域（包括 WD40 结构域）。 LRRK2 激酶活性增加与帕金森病的家族性和散发性相关，引起了人们对其细胞功能的强烈兴趣。然而，需要能够与 LRRK2 结合并报告或影响其细胞活性的小分子探针。在这里，我们报告了第一个高亲和力 LRRK2 结合设计的锚蛋白重复蛋白 (DARPin) 的鉴定和表征，名为 E11。使用冷冻电镜，我们证明 DARPin E11 与 LRRK2 WD40 结构域结合。与 DARPin E11 结合的 LRRK2 在冷冻电镜网格上表现出改善的行为，从而产生更高分辨率的 LRRK2 结构。 DARPin E11 不会影响截短形式 LRRK2 的催化活性，但会降低细​​胞中 LRRK2 底物 Rab8A 的磷酸化。我们还发现 DARPin E11 会破坏细胞中微管相关 LRRK2 丝的形成，而众所周知，这些丝需要基于 WD40 的二聚化。因此，DARPin E11 是探索 LRRK2 功能和功能障碍的新工具，并指导开发针对 WD40 结构域而不是激酶的 LRRK2 激酶抑制剂。