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. 2012;7(8):e43472.
doi: 10.1371/journal.pone.0043472. Epub 2012 Aug 29.

Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers

Laura Civiero  1 , Renée Vancraenenbroeck, Elisa Belluzzi, Alexandra Beilina, Evy Lobbestael, Lauran Reyniers, Fangye Gao, Ivan Micetic, Marc De Maeyer, Luigi Bubacco, Veerle Baekelandt, Mark R Cookson, Elisa Greggio, Jean-Marc Taymans
Affiliations

Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers

Laura Civiero et al. PLoS One. 2012.

Abstract

Leucine-rich repeat kinase 1 and 2 (LRRK1 and LRRK2) are large multidomain proteins containing kinase, GTPase and multiple protein-protein interaction domains, but only mutations in LRRK2 are linked to familial Parkinson's disease (PD). Independent studies suggest that LRRK2 exists in the cell as a complex compatible with the size of a dimer. However, whether this complex is truly a homodimer or a heterologous complex formed by monomeric LRRK2 with other proteins has not been definitively proven due to the limitations in obtaining highly pure proteins suitable for structural characterization. Here, we used stable expression of LRRK1 and LRRK2 in HEK293T cell lines to produce recombinant LRRK1 and LRRK2 proteins of greater than 90% purity. Both purified LRRKs are folded, with a predominantly alpha-helical secondary structure and are capable of binding GTP with similar affinity. Furthermore, recombinant LRRK2 exhibits robust autophosphorylation activity, phosphorylation of model peptides in vitro and ATP binding. In contrast, LRRK1 does not display significant autophosphorylation activity and fails to phosphorylate LRRK2 model substrates, although it does bind ATP. Using these biochemically validated proteins, we show that LRRK1 and LRRK2 are capable of forming homodimers as shown by single-particle transmission electron microscopy and immunogold labeling. These LRRK dimers display an elongated conformation with a mean particle size of 145 Å and 175 Å respectively, which is disrupted by addition of 6M guanidinium chloride. Immunogold staining revealed double-labeled particles also in the pathological LRRK2 mutant G2019S and artificial mutants disrupting GTPase and kinase activities, suggesting that point mutations do not hinder the dimeric conformation. Overall, our findings indicate for the first time that purified and active LRRK1 and LRRK2 can form dimers in their full-length conformation.

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Conflict of interest statement

Competing Interests: The authors have read the journal's policy and have the following conflicts: co-authors Elisa Greggio and Mark Cookson are Plos ONE Editorial Board members. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Characterization of HEK293T cell line stably expressing 3xFlag-LRRK1 and LRRK2.
(A) Schematic alignment of LRRK1 and LRRK2. Predicted functional domains are drawn to scale at the relative location within the full protein sequence. For domains containing repeat sequences, predicted individual repeat units are depicted. The sequence identity and similarity for the LRR, ROC, COR and Kinase domains are given below the schematic. Also given are detailed alignments of LRRK1 and LRRK2 at the level of common LRRK2 clinical mutations. Abbreviations for the domains: ARM, armadillo repeat domain; ANK, ankyrin repeat domain; LRR, leucine rich repeat domain; ROC, Ras of comple proteins domain; COR, C-terminal of ROC domain; Kin, kinase domain; WD40, WD40 repeat domain. (B) Representative western blot analysis of HEK293T cells stably expressing (from lane 1 to 7) 3xFlag-tagged LRRK2 wild-type, T1348N GTP deficient binding mutant, K1906M kinase dead, G2019S pathogenic mutant and LRRK1 wild-type K650A GTP deficient binding mutant, K1269M kinase dead. Upper panel shows membranes probed with Flag (M2) antibody (note that LRRK2 and LRRK1 have different exposure time due to the very low expression of T1348N mutant). Lower panel shows β-tubulin loading control. (C) Representative confocal images of stable HEK293T cells expressing LRRK1 and LRRK2 wild-type and mutants. Scale bar 10 µm.
Figure 2
Figure 2. Purification of soluble full-length 3xFlag-LRRK1 and 3xFlag-LRRK2.
(A) Representative silver staining of purified 3xFlag-LRRK1 and LRRK2 purification indicates highly pure protein fractions. Markers are in kilodaltons (B) Circular dichroism analysis of purified 3xFlag LRRK1 and LRRK2. Representative spectra are reported as mean residue molar ellipticity (deg cm2 dmol−1). (C) Representative fluorescence spectra of purified LRRK1 (right) and LRRK2 (left) before (solid line) and after (dashed line) addition of 6M GdHCl using an excitation wavelength of 280 nm. Fluorescence intensity was normalized to the highest peak.
Figure 3
Figure 3. LRRK1 and LRRK2 bind guanine nucleotides.
(A) Nucleotide competition assays with purified 3xFLAG-LRRK1/2 bound to M2-Flag affinity resin and incubated with a fixed concentration of GTP-α-P33 (10 nM) in the presence of 100 µM of cold nucleotides. Graph shows that loaded GTP- α-P33 is outcompeted by guanine nucleotides but not by ATP or CTP. (B) Nucleotide competition assays with proteins incubated with a fixed concentration of GTP- α-P33 (10 nM) and varying concentrations of cold GTP. Competition curves with GTP were used to generate IC50 values, which are apparent dissociation constants.
Figure 4
Figure 4. Comparative kinase activities of LRRK1 and LRRK2.
(A) Autophosphorylation assays of 3xFlag-LRRK1 wild-type, LRRK1 kinase dead, LRRK2 wild-type and LRRK2 kinase dead. End-point reactions (60 minutes) were resolved on a 4–20% SDS-PAGE and transferred onto PVDF membranes. Upper panel is autoradiography and lower panel western blot to correct activity for total loading (with anti-Flag antibody). The experiment is representative of n = 3 replicates. Markers to the right of the blots are in kilodaltons. (B) Quantitation of 33P signal by densitometry normalized to total loading. (C–D) LRRKtide (C) and (D) Nictide phosphorylation assessed by P81 filter binding assay reveals that both peptides are specific substrates for LRRK2. (E) Rate of P33 incorporation as a function of LRRK2 protein content (from 10 to 550 ng) measured by LRRKtide phosphorylation assays. (F) Kinetic constants of wild-type and G2019S LRRK2 for LRRKtide were determined by incubating 25 nM LRRK2 with varying concentrations of LRRKtide in the presence of 100 µM ATP and by fitting the data to a hyperbolic function. Km was 171±20 µM for wild-type and 257±63 µM for G2019S. Vmax were 1.92±0.06 pmol/min/µg for wild-type and 7.71±0.95 pmol/min/µg for G2019S. (G) ATP binding was tested for both LRRK1 and LRRK2 by affinity binding of the proteins to 4 different forms of ATP-bound agarose beads (ie ATP is coupled to the beads in different conformations) as described in materials and methods. Both LRRK1 and LRRK2 bound to the beads when ATP is coupled via the adenine moiety (6-AH-ATP-A or 8-AH-ATP-A). Binding was negligible for ATP coupled via the gamma-phosphate (AP-ATP-A) or ribose group (EDA-ATP-A). The position of the 250 kilodalton Mw marker is shown.
Figure 5
Figure 5. Analysis of LRRK1 and LRRK2 structure by transmission electron microscopy (TEM).
Distributions of gold particle distances of double-gold labeled LRRK1 (A) and LRRK2 (B) particles and representative images. Particles were stained with uranyl acetate and subsequently labeled with primary anti-Flag (M2) antibody and secondary 5 nm gold-labeled secondary anti-mouse antibody. (C and D) Distributions of particles diameters of purified LRRK1 and LRRK2 negative stained with uranyl acetate and representative images of protein shapes. (E) Scatter plots of gold distances measured for double-gold labeled LRRK1 and LRRK2. Distributions are significantly different as assessed by t-test (**, P = 0.001). (F) Scatter plots of particle size for LRRK1 and LRRK2. Distribution of LRRK1 and LRRK2 are significantly different by t-test (***, P<0.0001).
Figure 6
Figure 6. Analysis of different LRRK2 variants by immunogold EM reveals existence of dimeric proteins.
Distributions of gold particle distances of gold-labeled LRRK2 wild-type (A), G2019S pathological mutant (B), kinase dead K1906M (C) and GTP deficient binding mutant T1348N (D). Distances between particles were measured within 200 nm and weighted by the area of the annulus of thickness correspondent to the bin size (2.5 nm). See the materials and methods section for a more detailed explanation of the analysis.
Figure 7
Figure 7. Immunogold labeling of endogenous and 3xFlag-tagged ectopic LRRK2 chromatographic fractions from cell lysates.
(A) Relative chromatographic profiles built by measuring the dot blot immunoreactive signal intensity of each fraction and dividing it by the total signal of the protein in all fractions to determine the percentages shown in the graph. Endogenous LRRK2 from NIH-3T3 cells was detected using a rat monoclonal antibody , which recognizes endogenous LRRK2 as shown by western blot of a total lysate (inset image). (B) Representative TEM images of immunogold labeled samples from chromatographic fractions corresponding to 12.5 mL peaks. 3xFlag-LRRK2 was labeled using monoclonal M2 anti-flag antibodies and anti-mouse secondary antibodies conjugated with 5 nm gold particles; endogenous LRRK2 from NIH-3T3 cells was labeled using a anti-LRRK2 rabbit monoclonal antibody (aa 100–500) and anti-rabbit secondary antibodies conjugated with 10 nm gold particles. (C) Frequency distribution plots of gold-particle distances for 3xFlag-LRRK2 (black bins) and endogenous (red bins) chromatographic fractions analyzed by immunogold EM.
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