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Structure of LRRK2 in Parkinson’s disease and model for microtubule interaction

Nature volume 588, pages 344–349 (2020)Cite this article

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Abstract

Leucine-rich repeat kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinson’s disease1 and is also linked to its idiopathic form2. LRRK2 has been proposed to function in membrane trafficking3 and colocalizes with microtubules4. Despite the fundamental importance of LRRK2 for understanding and treating Parkinson’s disease, structural information on the enzyme is limited. Here we report the structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported cryo-electron tomography in situ structure5. We propose that the conformation of the LRRK2 kinase domain regulates its interactions with microtubules, with a closed conformation favouring oligomerization on microtubules. We show that the catalytic half of LRRK2 is sufficient for filament formation and blocks the motility of the microtubule-based motors kinesin 1 and cytoplasmic dynein 1 in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce the formation of LRRK2 filaments in cells, whereas inhibitors that stabilize a closed conformation do not. Our findings suggest that LRRK2 can act as a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

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Fig. 1: Cryo-EM structure of LRRK2RCKW.
Fig. 2: Modelling the microtubule-associated LRRK2 filaments.
Fig. 3: LRRK2RCKW forms WD40- and COR-mediated dimers outside the filaments.
Fig. 4: LRRK2RCKW inhibits the motility of kinesin and dynein.
Fig. 5: Type II, but not type I, kinase inhibitors rescue kinesin and dynein motility and reduce LRRK2 filament formation in cells.

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Data availability

All reagents and data will be made available upon request. Model coordinates for the LRRK2RCKW structure are deposited in the PDB as follows: (1) PDB accession code 6VP6: LRRK2RCKW with the adjacent COR-B and WD40 domains (from the trimer) used to optimize residues at those interfaces during refinement in Rosetta, with GDP-Mg2+ bound; (2) PDB accession code 6VNO: the top 10 models for LRRK2RCKW without adjacent domains, with GDP-Mg2+ bound; (3) PDB accession code 6VP8: LRRK2RCKW with the adjacent COR-B and WD40 domains (from the trimer) used to optimize residues at those interfaces during refinement in Rosetta, no GDP-Mg2+; (4) PDB accession code 6VP7: the top 10 models for LRRK2RCKW without adjacent domains, no GDP-Mg2+ bound. Cryo-EM maps for the different LRRK2RCKW structures are deposited at the EMDB as follows: (1) Electron Microscopy Data Bank (EMDB) accession code EMD-21250: this deposition contains both the 3.5 Å map of LRRK2RCKW trimer (used to build the COR-B, kinase and WD40 domains) and the 3.8 Å map of the signal-subtracted LRRK2RCKW trimer (used to build the ROC and COR-A domains); (2) EMDB accession code EMD-21306: 8.1 Å map of LRRK2RCKW monomer; (3) EMD accession code 21309: 9.5 Å map of COR-mediated LRRK2RCKW dimer in the absence of kinase ligand (apo); (4) EMDB accession code EMD-21310: 13.4 Å map of WD40-mediated LRRK2RCKW dimer in the absence of kinase ligand (apo); (5) EMDB accession code EMD-21311: 9.0 Å map of COR-mediated LRRK2RCKW dimer in the presence of MLi-2; (6) EMDB accession code EMD-21312: 10.2 Å map of WD40-mediated LRRK2RCKW dimer in the presence of MLi-2. All other data that support the findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank S. Taylor for her role in initiating this collaborative work, which was partially supported by multi-investigator grants from the Michael J. Fox Foundation: grants: 11425 and 11425.02 (PI: S. Taylor) and 18321 (PIs: A.E.L. and S.L.R.-P.). We also thank the UC San Diego Cryo-EM Facility, the Nikon Imaging Center at UC San Diego, where the confocal microscopy was performed, the use of instruments at the Electron Imaging Center for NanoMachines supported by NIH (1S10RR23057, 1S10OD018111, and 1U24GM116792), NSF (DBI-1338135) and CNSI at UCLA; J. P. Gillies and A. Kendrick for technical support with protein purifications, and A. Dickey for feedback on the manuscript. C.K.D. was initially supported by the Molecular Biophysics Training Grant (NIH grant T32 GM008326) and subsequently by a Predoctoral Fellowship from the Visible Molecular Cell Consortium and Center for Trans-scale Structural Biology (UC San Diego). D.S. is supported by an A. P. Giannini Foundation postdoctoral fellowship. A.K.S. receives salary and support from the Ludwig Institute for Cancer Research. E.V. is supported by a NIH Director’s New Innovator Award DP2GM123494. S.L.R.-P. is an investigator of the Howard Hughes Medical institute and is also supported by R01GM121772. A.E.L. is supported by R01GM107214. S.K. is grateful for support from the SGC, a registered charity that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative EUbOPEN (agreement No 875510), Janssen, Merck KGaA, MSD, Ontario Ministry of Economic Development and Innovation, Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and the Wellcome, as well as Boehringer Ingelheim for funding initial structural studies of this project. Most of this work is described in the thesis67 by C.K.D.

Author information

Author notes

  1. C. K. Deniston

    Present address: Genomics Institute of the Novartis Research Foundation, La Jolla, CA, USA

  2. I. Lahiri

    Present address: Department of Biological Sciences, Indian Institute of Science Education and Research Mohali, Mohali, India

  3. R. Watanabe

    Present address: La Jolla Institute for Immunology, La Jolla, CA, USA

  4. J. Böhning

    Present address: Sir William Dunn School of Pathology, Oxford University, Oxford, UK

  5. These authors contributed equally: C. K. Deniston, J. Salogiannis, S. Mathea

Authors and Affiliations

  1. Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA

    C. K. Deniston, J. Salogiannis, D. M. Snead, I. Lahiri, M. Matyszewski, S. L. Reck-Peterson & A. E. Leschziner

  2. Howard Hughes Medical Institute, Chevy Chase, MD, USA

    J. Salogiannis, O. Donosa & S. L. Reck-Peterson

  3. Institute of Pharmaceutical Chemistry, Goethe-Universität, Frankfurt, Germany

    S. Mathea & S. Knapp

  4. Division of Biological Sciences, Molecular Biology Section, University of California San Diego, La Jolla, CA, USA

    R. Watanabe, J. Böhning, E. Villa & A. E. Leschziner

  5. Small Molecule Discovery Program, Ludwig Institute for Cancer Research, La Jolla, CA, USA

    A. K. Shiau

  6. Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, USA

    A. K. Shiau & S. L. Reck-Peterson

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Contributions

C.K.D. collected and processed the cryo-EM data. J.S. performed the single-molecule and cellular assays with the help of O.D. S.M. designed the LRRK2RCKW construct and purified the protein. C.K.D. and I.L. built the molecular model of LRRK2RCKW. D.M.S. performed the SEC–MALS and phosphorylation assays. M.M. collected and analysed the LRRK2RCKW and microtubule cryo-EM data. R.W. and J.B. performed the cellular cryo-ET. A.K.S. contributed to the structural analysis and provided advice on the selection of kinase inhibitors. S.K., E.V., S.L.R.-P. and A.E.L. directed and supervised the research. C.K.D., J.S., S.L.R.-P. and A.E.L. wrote the manuscript and S.M., D.S., M.M., O.D., A.K.S., S.K. and E.V. edited it.

Corresponding authors

Correspondence to S. L. Reck-Peterson or A. E. Leschziner.

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The authors declare no competing interests.

Additional information

Peer review information Nature thanks Asa Abelovich, Henning Stahlberg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 Optimization of LRRK2 constructs and cryo-EM analysis of a LRRK2RCKW trimer.

a, We systematically scanned domain boundaries (amino acid numbers of boundaries noted above domain names) to generate LRRK2 constructs that expressed well in baculovirus-infected insect cells and yielded stable and soluble protein. These attempts included full-length LRRK2, the kinase domain alone or with the WD40 domain, and other isolated domains. In this approach, only the GTPase domain on its own expressed well. Next, we gradually shortened LRRK2 from its amino terminus. Red asterisks indicate constructs that were soluble. b, After identifying domain boundaries yielding constructs that expressed soluble protein, additional fine tuning of boundaries was performed. A Coomassie-stained SDS–PAGE gel shows systematic N-terminal truncations at the ROC domain resulting in the identification of a construct with the highest expression levels: amino acids 1327–2527 (red asterisk, ‘LRRK2RCKW’ here). c, A Coomassie-stained SDS–PAGE gel of purified LRRK2RCKW after elution from an S200 gel filtration column. As predicted by its primary structure, LRRK2RCKW runs at approximately 140 kDa. d, Electron micrograph of LRRK2RCKW. e, 2D class averages of the LRRK2RCKW trimer. f, 2D/3D classification scheme used to obtain the 3.5 Å structure of the LRRK2RCKW trimer. g, h, Fourier shell correlations (from Cryosparc) (d) and Euler angle distribution (e) for the LRRK2RCKW trimer.

Extended Data Fig. 2 Cryo-EM analysis of a signal-subtracted LRRK2RCKW trimer and map-to-model fit.

a, Processing strategy used to obtain a 3.8 Å structure of LRRK2RCKW generated from a signal-subtracted trimer where only one monomer contains the ROC and COR-A domains. This structure improved the resolution of the ROC and COR-A domains relative to the full trimer (Extended Data Fig. 1). b–d, 2D class averages (b), Fourier shell correlations (from Relion) (c), and Euler angle distribution (from Relion) (d) for the 3.8 Å resolution signal-subtracted LRRK2RCKW structure. e, Close-ups (f–l) of different parts of the final model fit into the map. f, Section of the WD40 domain. g, C-terminal helix and its interface with the kinase domain. h, Active site of the kinase. Residues in the DYG motif are labelled. G2019, the site of a major PD-associated mutation (G2019S) and the last residue of the activation loop seen in our structure, is highlighted by a black rounded square. i, Interface between COR-B and the αC helix of the N-lobe of the kinase domain. j, Interface between the ROC and COR-B domains. R1441 and Y1699, two residues mutated in Parkinson’s disease, are labelled. k, l, Two different views of the ROC and COR-A domains with GDP-Mg2+ modelled into the density. Side chains were omitted in these two panels, corresponding to the lowest-resolution parts of the map. m, Map-to-model FSC plots for the top-ranked LRRK2RCKW models, with (left) or without (right) GDP-Mg2+ (right) in the ROC domain. The 0.143 FSC values are reported in Supplementary Table 1. n, Size exclusion chromatography–multiple angle light scattering (SEC–MALS) analysis of LRRK2RCKW under the conditions used for cryo-EM (Fig. 1). The table shows the calculated molecular weights (MW) of LRRK2RCKW according to SEC standards and MALS.

Extended Data Fig. 3 Comparisons between LRRK2 and other kinases and modelling of the LRR into LRRK2RCKW.

a, View of the LRRK2RCKW atomic model with COR-A, COR-B and kinase domains coloured. The N- and C-lobes of the kinase are labelled, as is the αC helix in the N-lobe. b, c, The FAK-FERM (PDB code 2J0J)17 (b) and CDK2-cyclin A (PDB code 2CCH)19 (c) complexes, shown in the same orientation as the kinase in a. The αC helix of CDK2 is also labelled. d, Same view as in a with only the kinase domain and the C-terminal helix coloured. e, Rotated view of the LRRK2 kinase domain with the C-terminal helix facing the viewer. f, g, CDKL3 (PDB code 3ZDU) (f) and RIPK2 (PDB code 4C8B)32 (g) shown in the same orientation as the LRRK2 kinase in e, with alpha helices with the same general location as the LRRK2 C-terminal helix coloured in green. h, KSR2-MEK1 complex (PDB code 2Y4I), with the kinase oriented as in e (left) and after removing KSR2 for clarity (right). The alpha helix associated with the kinase is shown in green. i, HCK (PDB code 2HCK) in complex with its SH2 and SH3 domains with the kinase oriented as in e (left), and after removal of the SH2 and SH3 domains for clarity (right). A remaining alpha helix from the SH2 domain is shown in yellow. j, Front view of the LRRK2 kinase with the C-spine and R-spine residues coloured in grey and white, respectively. k, Close-up of the DYG motif and neighbouring R-spine residues. A putative hydrogen bond between Y2018 and the backbone carbonyl of I1933 is shown (O–O distance: 2.7 Å). This interaction provides a structural explanation for the hyperactivation of the kinase resulting from a Y2018F mutation38, which would release the activation loop. l, Crystal structure of the LRR–ROC–COR(A/B) domains from C. tepidum Roco (PDB code 6HLU)7. m, Homology model for human LRR–ROC–COR(A/B) based on the C. tepidum Roco structure (from SWISS-MODEL). n, Chimeric model combining LRRK2RCKW and the homology model for the LRR domain from m obtained by aligning their ROC–COR(A/B) domains. o, p, Two views of the hybrid LRRK2LRCKW model. q, Close-up showing the proximity between the active site of the kinase (with the side chains of its DYG motif shown) and the S1292 autophosphorylation site on the LRR. The close-up also highlights the proximity between N2081, a residue implicated in Crohn’s disease, and the LRR.

Extended Data Fig. 4 Comparison between LRRK2RCKW and integrative models built into cryo-ET maps of LRRK2 filaments in cells and docking of LRRK2RCKW into those maps.

a, Root-mean-square deviation (r.m.s.d.) between the atomic model of LRRK2RCKW and each of the 1,167 integrative models previously generated5. r.m.s.d. values were calculated in Chimera62 using 100% residue similarity and with pruning iterations turned off. r.m.s.d. values are grouped into 53 clusters of related models (see ref. 5 for details), with the mean and standard deviation shown whenever the cluster contains two or more models. Integrative models that gave the lowest, median and highest r.m.s.d. values are shown. The models are coloured according to the per-residue r.m.s.d. with the atomic model of LRRK2RCKW. b, The WD40s in the crystal structure of a dimer of the LRRK2 WD40 (PDB code: 6DLP)9 were replaced with the WD40s from our cryo-EM structure of LRRK2RCKW. c, The resulting dimer was fitted into the 14 Å cryo-ET map of cellular microtubule-associated LRRK2 filaments5. d, Two views of the same fitting shown in c, displayed with a higher threshold for the map to highlight the fitting of the WD40 β-propellers into the density. The white arrows point towards the holes at the centre of the β-propellers densities. e, Four copies of LRRK2RCKW were docked into the cryo-ET map by aligning their WD40 domains to the docked WD40 dimer. f, Model containing the four aligned LRRK2RCKW. g–j, Modelling of the kinase-closed form of LRRK2RCKW. g, h, The structure of ITK bound to an inhibitor (PDB code 3QGY)63, which is in a closed conformation, was aligned to LRRK2RCKW using only the C-lobes of the two kinases. i, The N-terminal portion of LRRK2RCKW, comprising ROC, COR-A, COR-B and the N-lobe of the kinase, was aligned to ITK using only the N-lobes of the kinases. ROC, COR-A and COR-B were moved as a rigid body in this alignment. j, Kinase-closed model of LRRK2RCKW.

Extended Data Fig. 5 Ab initio models for cryo-EM of LRRK2RCKW dimers and cryo-EM analysis of WD40- and COR-mediated dimers of LRRK2RCKW in the presence of the inhibitor MLi-2.

a, An initial dataset was collected from a sample of LRRK2RCKW incubated in the presence of the kinase inhibitor MLi-2 and dimers were selected. b, Representative two-dimensional class averages used for ab initio model building. c, Ab initio models with the structure of LRRK2RCKW docked in. d, Volumes generated form the molecular models in b, filtered to 30 Å resolution. e, Projections of the volumes in d shown in the same order as their corresponding 2D class averages in b. f, Data processing strategy for obtaining cryo-EM structures of WD40- and COR-mediated dimers of LRRK2RCKW in the presence of the inhibitor MLi-2. The models used during this processing (Methods) are those shown in d along with an additional linear trimer (Methods) used for particle sorting.

Extended Data Fig. 6 Cryo-EM analysis of a monomer and WD40- and COR-mediated dimers of LRRK2RCKW in the absence of inhibitor (apo) and dimerization of LRRK2RCKW outside the filaments.

a, Data-processing strategy for obtaining cryo-EM structures of a monomer and WD40- and COR-mediated dimers of LRRK2RCKW in the absence of inhibitor. The models used during the processing of the dimers (Methods) are those shown in Extended Data Fig. 5d, along with an additional linear trimer (Methods) used for particle sorting. The models used for processing of the monomer (Methods) were the same dimer models as in Extended Data Fig. 5d (used for particle sorting) in addition to a monomer model generated from our LRRK2RCKW model (used for refinement). b, Two-dimensional (2D) class averages of WD40- and COR-mediated LRRK2RCKW dimers obtained in the absence of inhibitors (apo) or in the presence of either ponatinib or MLi-2. The same molecular models of the two dimers shown in Fig. 3 are shown on the left but in orientations similar to those represented by the 2D class averages shown here. For each class average, a projection from the corresponding model in the best-matching orientation is shown to its left. c, Two copies of the LRRK2RCKW structure were aligned to the ROC–COR domains of the LRR–ROC–COR structure from the C. tepidum Roco protein (PDB code 6HLU) to replicate the interface observed in the bacterial homologue in the context of the human protein. This panel shows a comparison between the dimer modelled based on the C. tepidum LRR–ROC–COR structure and the dimer observed for LRRK2RCKW in this work. Although the bacterial structure shows a dimerization interface that involves the GTPase (ROC), LRRK2RCKW interacts exclusively through its COR-A and -B domains, with the ROC domains located away from this interface. The two arrangements are shown schematically in cartoon form below the structures.

Extended Data Fig. 7 Properties of the microtubule-associated LRRK2RCKW filaments.

a, b, The LRRK2RCKW structure solved in this work (a) was split at the junction between the N- and C-lobes of the kinase domain (L1949-A1950) (b). c, Docking of the two halves of LRRK2RCKW into a cryo-EM map of a LRRK2RCKW dimer solved in the presence of MLi-2. The dimer map is the same one shown in Fig. 3 and Extended Data Figs. 10 and 11. d, The model obtained in c was docked into cryo-EM maps of either WD40- or COR-mediated dimers obtained in the presence of MLi-2. e, Molecular models resulting from the docking in d. f, Aligning, in alternating order, copies of the dimer models generated in d and e results in a right-handed filament with dimensions compatible with those of a microtubule, and its ROC domains pointing inwards (see Fig. 3g, h for more details). g, Docking of the two halves of LRRK2RCKW into a cryo-EM map of a LRRK2RCKW monomer solved in the absence of inhibitor (apo). The map is the one shown in Fig. 1g and Extended Data Fig. 6. h, Three-way comparison of LRRK2RCKW (with domain colours) and the models resulting from the dockings into the MLi-2 WD40-mediated dimer map (c) (dark blue) and apo monomer map (g) (light blue). The three structures were aligned using the C-lobes of their kinases and the WD40 domain. The superposition illustrates that the docking into the apo map results in a structure very similar to that obtained from the trimer (Fig. 1) and that the presence of MLi-2 leads to a closing of the kinase. i, Molecular model of the microtubule-associated LRRK2RCKW filament obtained by docking a fragment of a microtubule structure (PDB code 6O2S) into the corresponding density in the sub-tomogram average (Fig. 2a). j, Same view as in i with the models shown as surface representations coloured by their Coulomb potential. k, l, ‘Peeling off’ of the structure shown in j, with the LRRK2RCKW filament seen from the perspective of the microtubule surface (k) and the microtubule surface seen from the perspective of the LRRK2RCKW filament (l). Note that the acidic C-terminal tubulin tails are not ordered in the microtubule structure and are therefore not included in the surface charge distributions. The Coulomb potential colouring scale is shown on the right.

Extended Data Fig. 8 Inhibition of motor motility by wild-type and I2020T mutant LRRK2RCKW.

a, Example kymographs showing that increasing concentrations of LRRK2RCKW reduce kinesin runs. b, Example kymographs showing that 25 nM LRRK2RCKW reduces dynein runs. c, Representative kymographs of kinesin motility in the presence or absence of wild-type and I2020T mutant LRRK2RCKW. d, The percentage of motile kinesin events per microtubule in the absence of LRRK2 or in the presence of 25 nM wild-type or I2020T mutant LRRK2RCKW. Data are mean ± s.d. (n = 12 microtubules per condition quantified from two independent experiments). There is a significant difference between 0 nM and both 25 nM RCKW conditions (P < 0.0001), but no significant (ns) difference between the inhibitory effects of wild-type LRRK2RCKW versus I2020T mutant LRRK2RCKW as calculated using the Kruskal–Wallis test with Dunn’s posthoc for multiple comparisons (compared to no LRRK2RCKW).

Extended Data Fig. 9 Type II kinase inhibitors rescue kinesin and dynein motility.

a–e, Ponatinib is a type II, ‘DFG out’ inhibitor. a, Superposition of the structures of Ponatinib-bound RIPK2 (PDB code 4C8B)32 and IRAK4 (PDB code 6EG9). Ponatinib is shown in yellow, and the DYG motif residues are shown in white. b, c For comparison, the structures of Roco4 bound to LRRK2-IN-1 (PDB code 4YZM)35, a LRRK2-specific type I, ‘DFG in’ inhibitor (b), and a model of MAPK1 bound to MLi-2 (PDB code 5U6I)22, another LRRK2-specific type I, ‘DFG in’ inhibitor (c) are shown. The inhibitor and DFG residues are coloured as in a. d, The structures in a–c, as well as the kinase from LRRK2RCKW are shown superimposed. The colour arrowheads point to the N-lobe β-sheet to highlight the difference in conformation between kinases bound to the two different types of inhibitors. Note that the LRRK2RCKW kinase is even more open than the two ponatinib-bound kinases. e, Rotated view of d, now highlighting the position of the N-lobe αC helix. An additional alpha helix in the N-lobe of MAPK1 was removed from this view for clarity. f, The kinase inhibitors MLi-2 (1 μM), LRRK2-IN-1 (1 μM), ponatinib (10 μM) and GZD-824 (10 μM) all inhibit the LRRK2RCKW kinase activity in vitro compared to a DMSO control. A western blot using a phospho-specific antibody to Rab8A at the indicated time points is shown. g, A dose–response curve showing the percentage of motile kinesin events per microtubule as a function of ponatinib concentration with LRRK2RCKW (25 nM) or without LRRK2RCKW. Data are mean ± s.d. (from left to right: n = 12, 18, 16, 14 and 9 microtubules quantified from one experiment). ****P < 0.0001, Kruskal–Wallis test with Dunn’s posthoc for multiple comparisons, compared to DMSO without LRRK2RCKW. h, Dose–response curve of run lengths from data in g represented as a cumulative frequency distribution. From top to bottom: n = 654, 173, 584, 293 and 129 motile kinesin events. Mean decay constants (tau) ± confidence interval are (from top to bottom) 2.736 ± 0.113, 1.291 ± 0.181, 2.542 ± 0.124, 2.285 ± 0.134, and 1.653 ± 0.17. i, Representative kymographs of kinesin and dynein with DMSO or type II inhibitors with or without LRRK2RCKW. j, The type II kinase inhibitors ponatinib and GZD-824 rescue kinesin run length, represented as a cumulative frequency distribution of run lengths with LRRK2RCKW (25 nM) or without LRRK2RCKW. From top to bottom: n = 893, 355, 507, 499, 524 and 529 runs from two independent experiments. Mean decay constants (tau) ± 95% confidence intervals are (from top to bottom) 2.070 ± 0.058, 0.8466 ± 0.091, 1.938 ± 0.065, 2.075 ± 0.07, 1.898 ± 0.065 and 1.718 ± 0.064. Data were resampled with bootstrapping analysis and statistical significance was established using a one-way ANOVA with Dunnett’s test for multiple comparisons. DMSO run lengths were significantly different (P < 0.0001) between conditions (0 vs 25 nM RCKW). Ponatinib (0 vs 25 nM RCKW) and GZD-824 (0 vs 25 nM LRRK2) were not significant. k, As in j but with dynein. From top to bottom: n = 659, 28, 289, 306, 254 and 339 runs from two independent experiments. Mean decay constants (tau) ± 95% confidence intervals; micrometres are 4.980 ± 0.147, 0.846 ± 0.415, 4.686 ± 0.142, 4.445 ± 0.172, 3.156 ± 0.09, 3.432 ± 0.188 (from top to bottom). Statistical significance as in j and run lengths were significantly different (P < 0.0001) between DMSO conditions (0 vs 25 nM RCKW), and not significant for ponatinib or GZD0824 conditions. The DMSO conditions are reproduced from Fig. 4f for comparison. l, Expression levels of GFP-LRRK2 (I2020T) in 293T cells treated with either DMSO or GZD-824 (5 μM). An immunoblot with anti-GFP (LRRK2) and anti-GADPH (loading control), which is a representative image from three replicates, is shown. m, Quantification of GFP–LRRK2 (I2020T) expression levels from western blots similar to l. Data are mean ± s.d. (n = 3 per condition). GZD-824 is not significantly different from the DMSO-treated control (Mann–Whitney test). n, 293T cells immunostained for tubulin showing that the microtubule architecture is not affected by GZD-824 or MLi-2 compared to DMSO treatment. See Supplementary Table 1 for all source data and replicate information.

Extended Data Table 1 Cryo-EM data collection and model refinement statistics
Full size table

Supplementary information

Supplementary Figure 1

Western blots shown in Extended Data Fig. 9.

Reporting Summary

Supplementary Table 1

Data for statistics and replicate information

Video 1

| LRRK2RCKW Structure Overview of the cryo-EM structure of LRRK2RCKW with close-ups of its domains.

Video 2

| Parkinson’s Disease mutations in LRRK2RCKW Structure Close-ups of the residues most commonly mutated in PD. I2020 (mutated to T2020 in PD) is disordered in our structure and this is indicated by the square brackets around its name.

Video 3

| The C-terminal helix of LRRK2RCKW Close-up of the C-terminal helix of LRRK2RCKW showing the residues involved in electrostatic and hydrophobic interactions between it and the kinase domain.

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Deniston, C.K., Salogiannis, J., Mathea, S. et al. Structure of LRRK2 in Parkinson’s disease and model for microtubule interaction. Nature 588, 344–349 (2020). https://doi.org/10.1038/s41586-020-2673-2

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