Institute of Pharmaceutical Chemistry, Goethe-Universität, Frankfurt, Germany; Structural Genomics Consortium (SGC), Buchmann Institute for Life Sciences, Goethe-Universität, Frankfurt, Germany; Aligning Science Across Parkinson's (ASAP), Chevy Chase, Maryland, USA.
Aligning Science Across Parkinson's (ASAP), Chevy Chase, Maryland, USA; Department of Cellular and Molecular Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA; Department of Molecular Biology, School of Biological Sciences, University of California, San Diego, La Jolla, California, USA.
J Biol Chem. 2024 Jul;300(7):107469. doi: 10.1016/j.jbc.2024.107469. Epub 2024 Jun 12.
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.
富含亮氨酸重复激酶 2（LRRK2）是一种大型多功能蛋白，包含两个催化结构域、一个激酶和一个 GTPase，以及包括 WD40 结构域在内的蛋白相互作用结构域。LRRK2 激酶活性的增加与家族性和散发性帕金森病都有关联，这使得人们强烈关注其细胞功能。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）E11 的鉴定和表征。通过冷冻电镜，我们显示 DARPin E11 结合到 LRRK2 WD40 结构域。与 DARPin E11 结合的 LRRK2 在冷冻电镜网格上表现出更好的行为，从而获得更高分辨率的 LRRK2 结构。DARPin E11 不会影响 LRRK2 的截断形式在体外的催化活性，但会降低细胞中 LRRK2 底物 Rab8A 的磷酸化。我们还发现 DARPin E11 会破坏细胞中与微管相关的 LRRK2 纤维的形成，而这些纤维已知需要 WD40 基二聚化。因此，DARPin E11 是一种新的工具，可以探索 LRRK2 的功能和失调，并指导针对 WD40 结构域而不是激酶的 LRRK2 激酶抑制剂的开发。
