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. 2021 Jun 24;184(13):3519-3527.e10.
doi: 10.1016/j.cell.2021.05.004. Epub 2021 Jun 8.

Structural analysis of the full-length human LRRK2

Alexander Myasnikov  1 , Hanwen Zhu  2 , Patricia Hixson  2 , Boer Xie  3 , Kaiwen Yu  4 , Aaron Pitre  5 , Junmin Peng  6 , Ji Sun  7
Affiliations

Structural analysis of the full-length human LRRK2

Alexander Myasnikov et al. Cell. .

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are commonly implicated in the pathogenesis of both familial and sporadic Parkinson's disease (PD). LRRK2 regulates critical cellular processes at membranous organelles and forms microtubule-based pathogenic filaments, yet the molecular basis underlying these biological roles of LRRK2 remains largely enigmatic. Here, we determined high-resolution structures of full-length human LRRK2, revealing its architecture and key interdomain scaffolding elements for rationalizing disease-causing mutations. The kinase domain of LRRK2 is captured in an inactive state, a conformation also adopted by the most common PD-associated mutation, LRRK2G2019S. This conformation serves as a framework for structure-guided design of conformational specific inhibitors. We further determined the structure of COR-mediated LRRK2 dimers and found that single-point mutations at the dimer interface abolished pathogenic filamentation in cells. Overall, our study provides mechanistic insights into physiological and pathological roles of LRRK2 and establishes a structural template for future therapeutic intervention in PD.

Keywords: LRRK2; LRRK2 dimer; LRRK2 mutations; Parkinson's disease; kinase.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Structure of the LRRK2 monomer (A) SEC-MALS analysis of LRRK2 protein. The size-exclusion column WTC-030S5 (MW range 5,000–1,250,000 Da) was used to analyze the molecule weight of LRRK2. SEC-MALS data are plotted as a distribution of the weight average molecular weight (red) superimposed on the chromatogram of UV absorbance (black) at 280 nm as a function of elution volume. The final data show three peaks from left to right. Peak 1 is void, peak 2 is LRRK2 and Peak 3 contains contamination and degradation products of LRRK2. The SDS-PAGE of purified LRRK2 is also shown, and the band corresponding to LRRK2 is indicated by a red arrow. (B) The kinase activity of purified LRRK2. The kinase activity of LRRK2 is measured using an ADP-Glo™ assay. The y-axis is the bio-luminance signal. Data shown are the mean ± SD (n=4) (C) The overall structure of the LRRK2 monomer. Top: domain scheme of LRRK2. The domain boundary is mapped based on our cryoEM structure. The domain boundary is labeled. Bottom: cylinder model of the full-length LRRK2 at two views. ARM, ANK, LRR, ROC, COR, KIN and WD40 domains are colored in red, orange, yellow, green, cyan, blue and violet, respectively. The linkers connecting adjacent domains are colored using the same color as the previous domain. The same color code is used unless noted. See also Figures S1 and S2 and Table S1.
Figure 2.
Figure 2.
Inter-domain interaction and two scaffolding elements (A) Interdomain interaction between ROC and COR domains. PD disease mutation sites are shown as red spheres and labeled. (B) Interdomain interaction between KIN and ANK-LRR. The autophosphorylation site S1292 is shown as a red sphere. Sidechains of interacting residues between KIN and ANK-LRR are shown as ball-and-stick models, and mainchains are represented using spheres. Possible interactions are indicated by dashed lines. (C) The hinge helix connects the WD40, ANK and ARM domains. Residues that can be crosslinked are shown as spheres. K831-K756 is crosslinked in both our and previous studies (Guaitoli et al., 2016). The K831-K687 is a confident crosslinking pair in our study, and the K831-K2378 pair was reported with medium confidence. The disease mutation, G2385, facing R841 (shown as sticks and balls) of the hinge helix is colored in green. (D) The C-terminal helix interacts with KIN, ANK and COR domains. The side chains of E2527, R1866 and R1771 are shown as sticks and balls. Residues reported to be crosslinked with high confidence (K739-K2515 and K773-K2520) are shown as spheres. See also Figure S3 and Table S2.
Figure 3.
Figure 3.
The inactive kinase domain of LRRK2 and LRRK2G2019S. (A) Structural elements of the LRRK2 kinase domain. N-lobe and C-lobe are colored in blue and purple-blue and the “DYG” motif in magenta. The cryoEM density of the ATP molecule is shown. Interactions between Y2018 and K1906, Y2018 and E1920 are indicated by dashes. (B) The broken R-spine of the kinase domain. The four residues forming the R-spine (L1935, L1924, Y2018 and Y1992) are shown as green surfaces. (C) The position of KIN domain relative to ANK, LRR, ROC, COR and WD40 domains. (D) Interaction between KIN and LRR domains. (E) The kinase activity of LRRK2 and LRRK2G2019S. LRRK2G2019S shows ~1.4-fold increased kinase activity, compared to that of the wild-type LRRK2 (p<0.01, N=4). Data shown are the mean ± SD. (F) Comparison of kinase domains between the full-length LRRK2 (blue) and LRRK2G2019S (pink). ATP is shown as sticks. The densities of helices where the mutation is located are shown as grey meshes. See also Figure S3.
Figure 4.
Figure 4.
Structure of the LRRK2 dimer (A) Structure of LRRK2 dimers. The density and model of the well-resolved part of LRRK2 dimers are shown at two views. The two protomers are colored in orange and blue, respectively. (B) The dimer interface. The secondary structures of the COR-B subdomain are shown and numbered. Side chains of the interface residues are shown. (C) Imaging results of GFP-LRRK2RCKW wild type and Met1732Arg mutation in the presence and absence of MLi-2. The filaments are indicated by white arrows. The white scale bar is 10 µm in length. See also Figure S4 and Table S1.
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