Cryo-EM analysis of homodimeric full-length LRRK2 and LRRK1 protein complexes

International audience; Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD), and currently one of the most promising therapeutic targets for drug design in Parkinson's disease. In contrast, LRRK1, the closest homologue to LRRK2, does not play any role in PD. Here, we use cryo-electron microscopy (cryo-EM) and single particle analysis to gain structural insight into the full-length dimeric structures of LRRK2 and LRRK1. Differential scanning fluorimetry-based screening of purification buffers showed that elution of the purified LRRK2 protein in a high pH buffer is beneficial in obtaining high quality cryo-EM images. Next, analysis of the 3D maps generated from the cryo-EM data show 16 and 25 Å resolution structures of full length LRRK2 and LRRK1, respectively, revealing the overall shape of the dimers with twofold symmetric orientations of the protomers that is closely similar between the two proteins. These results suggest that dimerization mechanisms of both LRRKs are closely related and hence that specificities in functions of each LRRK are likely derived from LRRK2 and LRRK1's other biochemical functions. To our knowledge, this study is the first to provide 3D structural insights in LRRK2 and LRRK1 dimers in parallel. Parkinson's disease (PD) is the second most common neurodegenerative movement disorder. It affects 1–2% of all people above the age of 65 1 and is at present incurable, although treatments are available to alleviate the symptoms. Genetic studies have identified several genes involved in PD pathogenesis. The leucine rich repeat kinase 2 (LRRK2) gene is of particular importance, with mutations in the coding sequence being the most prevalent known causes of genetic PD and genomic variants at the LRRK2 locus being common risk factors for sporadic PD 2. In addition, LRRK2 appears to act upstream of several other PD genes and PD risk factors, such as alpha-synuclein, tau, cyclin G associated kinase (GAK) and RAB7L1 3, 4. The 144 kb-long LRRK2 gene encodes for the 2527 amino acids long, cytosolic enzyme LRRK2, which functions as a GTPase as well as a kinase. Most of the pathologically important mutations are clustered in the catalytic core of this protein, hinting that altered GTPase and kinase activities may play a crucial role in pathogenesis 5, 6. Targeting the LRRK2 signaling pathway is currently regarded as one of the most promising approaches in drug development for PD 7–10. LRRK2 is a member of the ROCO protein family 11. It contains several protein-protein interaction domains, including armadillo (ARM), ankyrin repeats (ANK), leucine-rich repeats (LRR), Ras Of Complex proteins GTPase (ROC), C-terminal Of ROC (COR), a kinase (KIN) and WD40 12 (Fig. 1). The multidomain protein is involved in several cellular functions, including autophagy and neurite outgrowth regulation, and is related to some mitochondrial diseases 13–15. Biochemical experiments suggest that the kinase and GTPase activities of LRRK2 are regulated by dimerization 16–19. Three other ROCO proteins have been identified in humans: Leucine-rich repeat kinase 1 (LRRK1), death-associated kinase 1 (DAPK1), and malignant fibrous histiocytoma amplified sequence 1 (MFASHI1). LRRK1 is the closest homologue of LRRK2. The domain organization is similar and, like LRRK2, LRRK1 is