PDF(Pubmed)
Abstract：
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 in vitro 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.
摘要：
富含亮氨酸的重复激酶2（LRRK2）是一种含有两个催化结构域的大型多域蛋白，一种激酶和一种GTP酶，以及蛋白质相互作用域，包括一个WD40域名。增加的LRRK2激酶活性与家族性和散发性帕金森病(PD)的关联引起了人们对确定其细胞功能的强烈兴趣。然而,需要能够结合LRRK2并报告或影响其细胞活性的小分子探针。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。使用低温EM，我们显示DARPinE11与LRRK2WD40结构域结合。与DARPinE11结合的LRRK2在低温EM网格上显示出改善的行为，导致更高分辨率的LRRK2结构。DARPinE11在体外不影响LRRK2截短形式的催化活性，但降低了Rab8A的磷酸化，LRRK2底物，在细胞中。我们还发现DARPinE11破坏细胞中微管相关LRRK2细丝的形成，已知需要基于WD40的二聚化。因此，DARPinE11是探索LRRK2的功能和功能障碍的新工具，并指导靶向WD40结构域而不是激酶的LRRK2激酶抑制剂的开发。
