DOI: 10.5281/zenodo.13837193
title: Supplemental data for "Intramolecular feedback regulation of the LRRK2 Roc G domain by a LRRK2 kinase dependent mechanism" (Gilsbach et al., eLife 2024, doi:10.7554/eLife.91083)

description: Supportive data for the eLife version of record.

(1) Data used for the Michaelis Menten Kinetics.

HPLC-based assay. Steady-state kinetic measurements of LRRK2-mediated GTP hydrolysis were performed as previously described (Ahmadian et al., 1997). Briefly, 0.1 µM of full-length LRRK2 was incubated with different amounts of GTP (0, 25, 75, 150, 250, 500, 1000, 2000, 3000 and 5000 µM) and production of GDP was monitored by reversed phase C18 HPLC. To this end, the samples (10 µl) were directly injected on a reversed-phase C18 column (pre-column: Hypersil Gold, 3µm particle size, 4.6x10mm; main column: Hypersil Gold, 5µm particle size, 4.6x250mm, Thermo Scientific) using an Ultimate 3000 HPLC system (Thermo Scientific, Waltham, MA, USA) in HPLC-buffer containing 50 mM KH2PO4/K2HPO4 pH 6.0, 10 mM tetrabutylammonium bromide and 10-15% acetonitrile. Subsequently, samples were analyzed using the HPLC integrator (Chromeleon 7.2, Thermo Scientific, Waltham, MA, USA). Initial rates of GDP production were plotted against the GTP concentration using GraFit5 (v.5.0.13, Erithacus Software). The number of experiments is indicated in the graph and data point is the average (±s.e.m.) of indicated repetitions. The Michaelis-Menten equation was fitted to determine KM (±s.e.) and kcat (±s.e.). Excel sheets used for the calculation of means are provided. No values are reported if the HPLC separation failed (e.g. unstable baseline).

Charcoal GTP hydrolysis assay. The [γ-32P]GTP charcoal assay was performed as previously described (Bollag and McCormick, 1995). Briefly, 0.1 µM full-length LRRK2 or 0.5 µM 6xHIS-MBP-RocCOR was incubated with different GTP concentrations, ranging from 75 µM to 8 mM, in the presence of [γ-32P] GTP in GTPase assay buffer (30 mM Tris pH 8, 150 mM NaCl, 10 mM MgCl2, 5% (v/v) Glycerol and 3 mM DTT). Samples were taken at different time-points and immediately quenched with 5% activated charcoal in 20 mM phosphoric acid. All non-hydrolyzed GTP and proteins were stripped by the activated charcoal and sedimented by centrifugation. The radioactivity of the isolated inorganic phosphates was then measured by scintillation counting. The initial rates of γ-phosphate release and the Michaelis-Menten kinetics were calculated as described above.

(2) Profile plots (Raw data) obtained for the Mass photometry analysis for T1343A vs WT LRRK2.

MP was performed as described in (Guaitoli et al., 2023). Briefly, the dimer ratio of LRRK2 was determined on a Refeyn Two MP instrument (Refeyn). Prior to the experiment, a standard curve relating particle contrasts to molecular weight was established using a Native molecular weight standard (Invitrogen, 1:200 dilution in HEPES-based elution buffer: 50 mM HEPES [pH 8.0], 150 mM NaCl supplemented with 200 µM desthiobiotin). Prior to mass photometry, the proteins, either WT or T1343A LRRK2, were incubated with ATP to induce autophophorylation. The LRRK2 protein was diluted to 2x of the final concentration (end concentrations: 75 nM and 100 nM) in elution buffer. The optical setup was focused in 10 μl elution buffer before adding 10 µl of the adjusted protein sample. Depending on the obtained count numbers, acquisition times were chosen between 20 s to 1 min. The dimer ratio in each measurement was normalize according to the equation. The measurement was perfomed in triplicates.

(3) AlphaFold3 model of LRRK2-pT1343 either bound to GDP/Mg or GTP/Mg.

Using AlphaFold3 (Abramson et al., 2024), we modeled and compared the GDP vs the GTP-state of phospho-T1343 LRRK2. Interestingly, the AlphaFold3 model suggests, that the phosphate group of the pT1343 residue is orientated inwards thereby substituting the gamma phosphate of the GTP in the GDP-bound state of LRRK2. This finding is in well agreement with MD simulations published recently (Stormer et al., 2023).