Conformation and dynamics of the kinase domain drive subcellular location and activation of LRRK2

Abstract

To explore how pathogenic mutations of the multidomain leucine-rich repeat kinase 2 (LRRK2) hijack its finely tuned activation process and drive Parkinson's disease (PD), we used a multitiered approach. Most mutations mimic Rab-mediated activation by "unleashing" kinase activity, and many, like the kinase inhibitor MLi-2, trap LRRK2 onto microtubules. Here we mimic activation by simply deleting the inhibitory N-terminal domains and then characterize conformational changes induced by MLi-2 and PD mutations. After confirming that LRRK2_RCKW retains full kinase activity, we used hydrogen-deuterium exchange mass spectrometry to capture breathing dynamics in the presence and absence of MLi-2. Solvent-accessible regions throughout the entire protein are reduced by MLi-2 binding. With molecular dynamics simulations, we created a dynamic portrait of LRRK2_RCKW and demonstrate the consequences of kinase domain mutations. Although all domains contribute to regulating kinase activity, the kinase domain, driven by the DYGψ motif, is the allosteric hub that drives LRRK2 regulation.

Keywords: Gaussian accelerated molecular dynamics; Parkinson’s disease; hydrogen-deuterium exchange mass spectrometry (HDX-MS); kinase regulation; leucine-rich repeat kinase 2 (LRRK2).

Fig. 1.

Schematic domain organization of LRRK2. Full-length protein (blue box) consists of the Armadillo domain (Arm), Ankyrin repeat (Ank), leucine-rich repeat, a GTPase domain called Ras of complex, C terminus of the Roc domain, kinase domain, and WD40 domain. The N-terminal domains contain the Arm, Ank, and LRR domains. The C-terminal domains, corresponding to the LRRK2_RCKW construct (red box), contain the ROC, COR, kinase, and WD40 domains.

Fig. 2.

Localization of the LRRK2-G2019S mutant and LRRK2_RCKW variants. (A) Time-lapse imaging of HEK293T cells transiently expressing YFP-LRRK2-G2019S: Confocal images (YFP fluorescence signal, maximum-intensity projections) were acquired every 11 min. Representative images show the typical diffuse cellular localization of the proteins (t = 0 h) prior to treatment with 100 nM MLi-2; following MLi-2 addition, proteins relocalize to form cytoplasmic filamentous structures (yellow arrows; +MLi-2, t = 2.5 h). After washout of the inhibitor, the proteins gradually dissociate from the filaments into the cytosol (washout; t = 2 to 3 h). (B) Time-lapse imaging of HEK293T cells transiently expressing YFP-LRRK2-G2019S before (t = 0 h) and after treatment with 100 nM rebastinib. No changes in the localization of the proteins are observed. (Scale bar, 20 μm.) (C) Plasmids, encoding LRRK2_RCKW variants, were transfected into HEK293T cells for LRRK2_RCKW overexpression. Transfected cells were then analyzed for the spatial distribution of LRRK2_RCKW by immunostaining. All tested LRRK2_RCKW variants displayed a high likelihood (80 to 90%) of forming filaments inside the HEK293T cells except for LRRK2_RCKW D2017A (20 to 30%). Interestingly, in contrast to full-length LRRK2, the percentage of cells showing filament formation was independent of MLi-2 treatment or a specific LRRK2_RCKW mutation. The figures show the filaments of WT and G2019S. The figures of other LRRK2_RCKW variants are shown in SI Appendix, Fig. S2. Error bars represent standard deviations (SD) for the percentage of filament forming cells on six to ten representative images taken per transfection (n = 2).

Fig. 3.

LRRK2_RCKW variants Y2018F and G2019S enhance LRRKtide and Rab8a phosphorylation. (A) An LRRKtide-based kinase assay for LRRK2_RCKW variants revealed that it preserves full-length LRRK2 kinase activity. Additionally, the DYGψ mutants tested here also resemble the results of their full-length counterparts. Interestingly, also the pathogenic mutations R1441C and Y1699C which are situated in the ROC–COR region of the LRRK2_RCKW construct display a mild increase in kinase activity compared with LRRK2_RCKW WT. Asterisks indicate the P value by one-way ANOVA test: 0.01 < *P < 0.05; 0.001 < **P < 0.01; ****P < 0.0001. Error bars represent SD for at least five independent measurements. (B) When testing Rab8a as a substrate for the LRRK2_RCKW construct employing Western blotting against pT72 and the His tag of His-Rab8a, we revealed increased phosphorylation of Rab8a by LRRK2_RCKW Y2018F, G2019S, and Y1699C. MLi-2 was shown to efficiently block phosphorylation of Rab8a which was also found for the kinase-dead mutant D2017A. Quantification was performed for three independent Western blots. For each quantification, the pT72 signals were referenced to the signal for the His tag of 6×His-Rab8a and then normalized to the resulting WT signal. The dotted line therefore represents 100% of the WT signal. Error bars represent SD of the quantification of three independent Western blots.

Fig. 4.

Deuterium uptake of the LRRK2_RCKW kinase domain. (A) The relative deuterium uptake after 2 min of deuterium exposure of the LRRK2_RCKW kinase domain is shown in a color-coded homology model. Gray color indicates no deuterium uptake information. The N lobe of the kinase mostly shows blue to green colors indicating low deuterium uptake. On the other hand, the αD-helix, activation loop, and end of αF-helix to αH-helix have higher deuterium uptake suggesting a more dynamic, solvent-accessible C lobe. (B) Representative peptides that have almost no deuterium uptake are mapped on the kinase domain. (B, Insets) Uptake for the apo kinase (black) and the MLi-2–bound state (red).

Fig. 5.

Binding of MLi-2 reduces the deuterium uptake of LRRK2_RCKW. (A) The relative deuterium exchange for each peptide detected from the N to C terminus of LRRK2_RCKW in apo kinase (black) and MLi-2–bound (red) conditions at 2 min. The arrows indicate the regions of LRRK2 that have less deuterium uptake when bound to MLi-2. (B) The deuterium uptake of selected peptides is plotted and mapped on the kinase model. The uptake is reduced in the Gly-rich loop, αC-helix, activation loop, DYGI motif, YRD motif, and hinge region in the presence of MLi-2.

Fig. 6.

Deuterium uptake and spectral plot of peptides in the DYGI (A), activation loop (B), and αC-helix (C) reveal slow dynamics. In the DYGI peptide (amino acids 2013 to 2022) the apo state (black) plateaus within 2 min. The MLi-2–bound state (red) continues to slowly exchange at least up to 5 min, suggesting that with MLi-2 this region undergoes a slow dynamic process. The apo state of the activation-loop peptide (amino acids 2028 to 2056) again plateaus within 2 min while the MLi-2–bound state gradually increases after 2 min. From the spectral plot, the uptake of the activation-loop peptide in the MLi-2 state exhibits bimodal behavior. One process has slow deuterium uptake (protected) and the other process has fast uptake (solvent-exposed)—similar to the single process observed in the apo state. For the αC-peptide (amino acids 1915 to 1921), the deuterium increases without reaching a plateau over 5 min for both states.

Fig. 7.

Mutations in the DYGψ loop alter kinase dynamics. (A) Kinase conformational free-energy landscape, represented by “open–close”: the distance from the top (K1906/β3-sheet) to the bottom (D1994/YRD motif) of the active site; and “αC in–out”: the distance between K1906 and E1920/αC-helix. The white line shows the closed-active kinase conformation. WT samples the active state infrequently, whereas the mutants more readily access the closed-active conformation. However, D2017A is destabilized to a more open conformation. PMF, potential of mean force. (B) In WT, Y2018 is locked in an inactive orientation by hydrogen bonds with I2015 and I1933. Y2018F packs with L1924 and releases the DYGI loop from an inactive state helping to assemble the active site. Y2018F breaks the interaction leading to increased side-chain dynamics, measured by the distance between the 2018 ζ-carbon and the backbone of I1933. (C) I2020T makes a hydrogen bond with the backbone of Y1992, coupling the DYGI and catalytic loops, which decreased backbone dynamics. The mutation brings the DYGI and YRD motifs together, measured as the distance from the 2020 Cβ and the backbone of Y1992. (D) G2019S bridges the DYGI loop to the αC-helix and β3-sheet, through E1920 and K1906. This stabilizes the DYGI loop, shown by rmsd, and promotes the closed kinase conformation.

Fig. 8.

Y2018 introduces strain into the DYGI loop. (A) The WT Y2018 side chain is in a high-energy conformation (magenta circle), illustrated by the side-chain torsion of Y2018 overlaid with the ideal tyrosine χ1/χ2 gauche(-) torsions (45), shown with increasing torsion preference (blue to red), whereas Y2018F (white circle) relieves the nonideal conformation. The χ1/χ2 side-chain torsion angle distribution for Y2018 and Y2018F during the simulations demonstrates the WT deviation from ideal space (Right). The white dashed lines indicate the preferred χ1 for Tyr. (B) The consequence of the nonpreferred side-chain conformation in WT manifests as strain in the backbone conformation, illustrated by the probability distribution of the Tyr ψ-dihedral angle. The ideal backbone conformation for the Tyr gauche(-) rotamer (45) is depicted in blue. The nonpreferred Y2018 rotamer in WT adds strain to the backbone (black area; arrows indicate nonideal backbone conformations). Y2018F populates ideal dihedral space (red area). The φ/ψ distribution of the Y/F2018 backbone during the simulation is shown (Right). The WT Y2018 backbone is more disordered than Y2018F. The mutation stabilizes the entire DYGI backbone as also illustrated in the conformational ensemble from MD (A).

Fig. 9.

Impaired LRRK2 activation results in microtubule association. (A) In the mutated or MLi-2–inhibited state the autoinhibitory site and the NTDs get displaced already in the cytosol, and the kinase domain is in a constitutively active conformation. This short circuits the activation process which depends on Rab29 association. The active kinase conformation is sufficient to induce dimerization and thereby multimerization of LRRK2 around MTs. The LRRK2 multimers on the MT results in the filament formation phenotype. (B) The importance of the NTDs for stabilizing LRRK2 in an inactive cytosolically distributed state was tested by deleting the N terminus. The resulting LRRK2_RCKW deletion construct spontaneously forms filaments around MTs and is no longer able to become recruited by Rab29 as it lacks the interaction sites in the Arm and/or Ank domain. We believe that the missing AI domain, which we think is positioned in the NTDs, forces the kinase domain of the RCKW construct into an active conformation resulting in multimerization and docking to MTs.