A designed ankyrin-repeat protein that targets Parkinson's disease-associated LRRK2

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.

Keywords: DARPin; LRRK2; Parkinson’s disease; Rab8a; WD40; cryo-electron microscopy; kinase; kinase inhibitor; microtubule.

Figure 1

DARPin E11 binds to LRRK2^RCKWwith high affinity. A, domain architecture of the LRRK2 protein. The same color scheme is used in all figures. The first residue of each domain is indicated. B and C, surface plasmon resonance analysis of DARPin E11 binding to LRRK2^RCKW. Recombinant LRRK2^RCKW was immobilized on a sensor chip and varying concentrations of DARPin E11 were added in the mobile phase. Sensograms are shown (no fit or errors were applied) (B) and the plateau values from the dose-response curves (C) are shown and fit to the Langmuir equation according to the least square method. A dissociation constant (K_D) of 70 nM ± 11 nM (SEM) was determined. D, the LRRK2^RCKW:E11 complex was subjected to size exclusion chromatography (SEC). Elution fractions were analyzed by SDS PAGE and visualized by Coomassie staining. The co-elution of LRRK2^RCKW and DARPin E11 showed that the complexes were stable during the SEC run. Molecular weight markers are noted on the left and the molecular weights of LRRK2^RCKW and DARPin E11 on the right. E, analysis of the LRRK2^RCKW:E11 complex by mass photometry. The molecular weight obtained from the yellow peak corresponds to that of a 1:1 LRRK2^RCKW:E11 complex. The tabular data for this figure can be found at: https://doi.org/10.5281/zenodo.10471514. DARPin, designed ankyrin-repeat protein; LRRK2, leucine rich repeat kinase 2.

Figure 2

Cryo-EM structure of the LRRK2^RCKW:DARPin E11 complex.A, Cryo-EM map (left), model (middle), and schematic representation (right) of LRRK2^RCKW bound to DARPin E11. Because of the focused refinement strategy used to maximize the resolution of the DARPin-WD40 part of the structure, only the kinase C-lobe and the WD40 domain are seen in this map. E11 binds to one of the faces of the WD40 domain, opposite the kinase active site, next to the central WD40 cavity. The eyes and arrows surrounding the rectangle on the model indicate the direction of the views shown in panels (C–E). B, surface representation of hydrophobicity of LRRK2^KW bound to DARPin E11, displaying the latter as a model. The most polar residues are shown in blue, and the most hydrophobic residues are shown in gold. C–E, close-up views of the binding interface. Key residues for the interaction are highlighted. F and G, Cryo-EM maps, FSC plots, and model-to-map fits for LRRK2^RCKW alone or bound to DARPin E11. H and I, plots of Euler angle distribution generated in cryoSPARC (left) or Relion (right) for LRRK2^RCKW alone (top) or bound to DARPin E11 (bottom). Addition of E11 increased the number of orientations adopted by LRRK2^RCKW on the cryo-EM grids. DARPin, designed ankyrin-repeat protein; FSC, fourier shell correlation; LRRK2, leucine rich repeat kinase 2.

Figure 3

DARPin E11 does not affect the kinase activity of LRRK2^RCKWin vitro. A, in vitro assay measuring the level of Rab8A phosphorylation by LRRK2^RCKW. Rab8A and LRRK2^RCKW were incubated in the presence of ATP and kinase activity was monitored by measuring the levels of substrate (Rab8A) and product (pRab8A) by mass spectrometry. B, Kinase activity of LRRK2^RCKW in the presence of either DARPin E11 (left) or the LRRK2-specific kinase inhibitor GNE-9605 (right). The IC₅₀ was 38 nM ± 3 nM (SD). Plot points represent three technical replicates. DARPin, designed ankyrin-repeat protein; LRRK2, leucine rich repeat kinase 2.

Figure 4

DARPin E11 disrupts LRRK2^FLfilament formation and decreases Rab8 phosphorylationin cells.A, modeling of E11 bound to the autoinhibited conformation of LRRK2^FL shows no steric clashes between E11 and LRRK2. B, the binding site of DARPin E11 on the WD40 domain overlaps with the WD40:WD40 dimerization interface that is involved in the formation of microtubule associated LRRK2 filaments. C, Rab8 phosphorylation in 293T cells overexpressing LRRK2^FL and GFP-Rab8, with or without DARPin E11-3xFLAG. 293T cells were transiently cotransfected with LRRK2^FL and GFP-Rab8 or LRRK2^FL, GFP-Rab8, and DARPin E11-3xFLAG for 48 h. Cells transfected only with LRRK2^FL and GFP-Rab8 were treated with DMSO or 2 μM MLi-2 for 1 h. Cells were lysed and immunoblotted for phospho-Rab8 (pT72), total GFP-Rab8, total LRRK2, DARPin E11-3xFLAG, and GAPDH. MW; molecular weight marker. D, quantification from five Western blots plotting the ratio of GFP-pRab8 to total GFP-Rab8. Plot points represent four technical replicates. Statistics were generated in GraphPad using a one-way ANOVA analysis with a Tukey’s multiple comparison of means. ∗∗∗∗p <0.0001 DMSO and MLi-2, ∗∗p = 0.0013 DMSO and DARPin E11, ∗∗p= 0.0012 MLi-2 and DARPin E11. E and F, representative images of 293T cells expressing either GFP-LRRK2^FL (E) or GFP-LRRK2^FL and DARPin E11 (F), treated with DMSO or MLi-2 for 2 h. G, quantification of the percent cells (mean ± sd) with GFP-LRRK2^FL filaments in the presence or absence or DARPin E11. Each data point on the graph represents a technical replicate. Three or four independent replicates were done per condition, with each replicate containing 48 to 140 cells. Statistics were generated using an one Way ANOVA with a Tuckey’s multiple comparison of means. ∗∗∗∗ p < 0.0001. ns, not significant. The tabular data for this figure can also be accessed at https://doi.org/10.5281/zenodo.10530220. DARPin, designed ankyrin-repeat protein; DMSO, dimethylsulfoxide; LRRK2, leucine rich repeat kinase 2.