Figures and data
Determination of kinetical parameters for LRRK2 GTP hydrolysis of PD variants by the Charcoal Assay. (A) Michaelis-Menten kinetics for pathogenic variant within the RocCOR module. (B) comparison of KM values. (C) Comparison of kcat values. (D) catalytic efficiency (kcat/KM). (E)-(G) Determination of kobs values for full-length LRRK2 at 100µM (E) and 2000µM (F) GTP. (H) Domain structure of LRRK2 and the position of PD variants analyzed in this study.
HPLC-based full-length LRRK2 Michaelis-Menten kinetics
Comparison of PD mutants in the HPLC-based GTPase assay for the LRRK2 full-length protein. (A) Michaelis-Menten kinetics for the R1441G Roc-domain variant compared to LRRK2 wt. (B) Michaelis-Menten kinetics for the G2019S kinase-domain variant compared to LRRK2 wt. (C) Michaelis-Menten kinetics for a kinase-dead variant compared to LRRK2 wt.
Kinetic parameters upon ATP incubation/ Identification of T1343 as relevant autophosphorylation site for a negative feedback loop. (A) Michaelis-Menten kinetics for LRRK2 wt +/− ATP, (B) Phospho-site screen: Position of the LRRK2 phosho-sites within the Roc domain which were included in the screen mapped on PDB:7LHW (Myasnikov et al., 2021). Individual domains are highlighted in color as follows: Armadillo (gray), Ankyrin (green), LRR (yellow), Roc (magenta), COR (wheat), Kinase (blue), WD40 (dark green). (C) Michaelis-Menten kinetics for T1343A LRRK2 +/− ATP.
Overview of the kinetical parameters for fl. LRRK2 GTPase determined by the HPLC assay. (A) KM values. (B) kcat values and (C) catalytic efficiency (kcat/KM). Significant differences have been determined by an ANOVA followed by a post hoc test (p=0.05: *).
Autophosphorylation-induced LRRK2 monomerization as shown in Guaitoli et al., 2023 (Guaitoli et al., 2023) is abolished by the T1343A variant. (A) Mass photometry assays for LRRK2 wt and (B) T1343A LRRK2. Significance has been determined by a student’s T-test (p=0.05: *; p=0.01: **).
Effect of Roc T1343A on LRRK2 kinase activity and comparison to PD variants (A) In vitro LRRKtide HPLC-based kinase assay. (B) Western blot for LRRK2 pS935, total LRRK2, Rab10 pT73 and total Rab10. (C) Relative Rab phosphorylation levels. (D) Relative LRRK2 pS935 levels. Significant differences have been determined by an ANOVA followed by a post hoc test (p=0.05: *).
MBP-RocCOR Michaelis-Menten kinetics as measured by charcoal based GTPase assay.
GTPase activity (kobs) as measured by charcoal based GTPase assay.
Cell-based phospho-Rab assays. Quantification of Western Blots
Michaelis-Menten kinetics for MLi-2 treated LRRK2 wt.
Initial Phosphosite-screen (alanine screen). Michaelis-Menten parameters were determined in dependence of ATP pre-incubation. (A) KM values. (B) kcat values. The number of replicates is indicated.
Raw data for the In vitro LRRKtide HPLC Assay (determination of kobs values). (A) LRRK2 G2019S, (B) LRRK2 wt, (C) LRRK2 T1343A, (D) kinase-dead (KD) LRRK2 K1906M.
Cell-based phosphor-Rab assays, blot raw images used for quantification. (A-C) biological replicates.
