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@MISC{Gilsbach:276097,
      author       = {Gilsbach, Bernd and Ho, Franz Y. and Zhang, Xiaojuan and
                      Kortholt, Arjan and Gloeckner, Christian Johannes},
      title        = {{D}ataset: {S}upplemental data for '{I}ntramolecular
                      feedback regulation of the {LRRK}2 {R}oc {G} domain by a
                      {LRRK}2 kinase dependent mechanism' ({G}ilsbach et al.,
                      e{L}ife 2024, doi:10.7554/e{L}ife.91083), v3},
      publisher    = {Zenodo},
      reportid     = {DZNE-2025-00178},
      year         = {2024},
      abstract     = {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 0.5
                      mM ATP or buffer (control) for 30 min at 30 ℃. 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). (4) Western blot RAW files for the cell-based phospho
                      Rab asssay (RAW data for Figure 6 supplement 2/ Supplemental
                      Figure 4 in the preprint version, Gilsbach et al, 2024)
                      Cell-based LRRK2 activity assays were performed as
                      previously described (Singh et al., 2022). Briefly, HEK293T
                      cells were cultured in DMEM (supplemented with $10\%$ Fetal
                      Bovine Serum and $0.5\%$ Pen/Strep). For the assay, the
                      cells were seeded onto six-well plates and transfected at a
                      confluency of $50-70\%$ with SF-tagged LRRK2 variants using
                      PEI-based lipofection. After 48 hours cells were lysed in
                      lysis buffer [30 mM Tris-HCl (pH7.4), 150 mM NaCl, $1\%$
                      NonidentP-40 substitute, complete protease inhibitor
                      cocktail, PhosStop phosphatase inhibitors (Roche)]. Lysates
                      were cleared by centrifugation at 10,000 x g and adjusted to
                      a protein concentration of 1 µg/µl in 1x Laemmli Buffer.
                      Samples were subsequently subjected to SDS PAGE and Western
                      Blot analysis to determine LRRK2 pS935 and Rab10 T73
                      phosphorylation levels, as described below. Total LRRK2 and
                      Rab10 levels were determined as a reference for
                      normalization. For Western blot analysis, protein samples
                      were separated by SDS–PAGE using NuPAGE $10\%$ Bis-Tris
                      gels (Invitrogen) and transferred onto PVDF membranes
                      (Thermo Fisher). To allow simultaneous probing for LRRK2 on
                      the one hand and Rab10 on the other hand, membranes were cut
                      horizontally at the 140 kDa MW marker band. After blocking
                      non-specific binding sites with $5\%$ non-fat dry milk in
                      TBST (1 h, RT) (25 mM Tris, pH 7.4, 150 mM NaCl, $0.1\%$
                      Tween-20), membranes were incubated overnight at 4°C with
                      primary antibodies at dilutions specified below.
                      Phospho-specific antibodies were diluted in TBST/ $5\%$ BSA
                      (Roth GmbH). Non-phospho-specific antibodies were diluted in
                      TBST/ $5\%$ non-fat dry milk powder (BioRad). Phospho-Rab10
                      levels were determined by the site-specific rabbit
                      monoclonal antibody anti-pRAB10(pT73) (Abcam, ab230261) and
                      LRRK2 pS935 was determined by the site-specific rabbit
                      monoclonal antibody UDD2 (Abcam, ab133450), both at a
                      dilution of 1:2,000. Total LRRK2 levels were determined by
                      the in-house rat monoclonal antibody anti-pan-LRRK2 (clone
                      24D8; 1:10,000) (Carrion et al., 2017). Total Rab10 levels
                      were determined by the rabbit monoclonal antibody
                      anti-RAB10/ERP13424 (Abcam, ab181367) at a dilution of
                      1:5,000. For detection, goat anti-rat IgG or anti-rabbit IgG
                      HRP-coupled secondary antibodies (Jackson ImmunoResearch)
                      were used at a dilution of 1:15,000 in TBST/ $5\%$ non-fat
                      dry milk powder. Antibody–antigen complexes were
                      visualized using the ECL plus chemiluminescence detection
                      system (GE Healthcare) using the Stella imaging system
                      (Raytest) for detection and quantification. Figure 6 Source
                      Data 1: Images generated by the Stella system are shown
                      which were used for quantification. The annotation file
                      equals Figure6-figure supplement 2 (Gilsbach et al., eLife
                      2024, doi:10.7554/eLife.91083). The lines corresponding to
                      LRRK2 pS935, total LRRK2, Rab10 pT73 and total Rab10 were
                      used for the quantification shown in Figure 6.},
      keywords     = {structural modeling (Other) / LRRK2 (Other)},
      cin          = {AG Gloeckner},
      cid          = {I:(DE-2719)1210007},
      pnm          = {352 - Disease Mechanisms (POF4-352)},
      pid          = {G:(DE-HGF)POF4-352},
      typ          = {PUB:(DE-HGF)32},
      doi          = {10.5281/ZENODO.14055918},
      url          = {https://pub.dzne.de/record/276097},
}