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)

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.本文对应eLife正式发表版本的支撑数据。 (1) 米氏动力学（Michaelis Menten Kinetics）相关实验数据 基于高效液相色谱（High Performance Liquid Chromatography, HPLC）的检测实验：LRRK2介导的GTP水解稳态动力学检测方法参照已发表文献（Ahmadian等，1997）进行。简要实验流程如下：取0.1 μM全长LRRK2蛋白，与不同浓度梯度的GTP（0、25、75、150、250、500、1000、2000、3000及5000 μM）共同孵育，采用反相C18 HPLC监测GDP生成量。具体操作：取10 μL样品直接注入反相C18色谱柱（预柱：Hypersil Gold，粒径3 μm，规格4.6×10 mm；分析柱：Hypersil Gold，粒径5 μm，规格4.6×250 mm，Thermo Scientific），使用Ultimate 3000 HPLC系统（Thermo Scientific，美国马萨诸塞州沃尔瑟姆市），流动相为含50 mM KH2PO4/K2HPO4（pH 6.0）、10 mM四丁基溴化铵及10%~15%乙腈的HPLC缓冲液。随后采用HPLC积分系统（Chromeleon 7.2，Thermo Scientific，美国马萨诸塞州沃尔瑟姆市）对样品进行分析。使用GraFit5（v.5.0.13，Erithacus Software）将GDP生成的初始速率对GTP浓度作图，通过拟合米氏方程求得米氏常数KM（±标准误）及催化常数kcat（±标准误）。实验次数如图中所示，数据点为指定重复次数实验的平均值（±均值标准误，±s.e.m.）。本研究提供用于计算平均值的Excel表格文件。若HPLC分离失败（如基线不稳定），则不给出对应数值。 活性炭吸附法GTP水解检测实验：[γ-32P]GTP活性炭吸附实验参照已发表文献（Bollag与McCormick，1995）进行。简要流程如下：取0.1 μM全长LRRK2蛋白或0.5 μM 6xHIS-MBP-RocCOR蛋白，在含[γ-32P]GTP的GTP酶检测缓冲液（30 mM Tris pH 8、150 mM NaCl、10 mM MgCl2、5%（v/v）甘油及3 mM DTT）中，与75 μM至8 mM的不同浓度梯度GTP共同孵育。在不同时间点取样，立即用含5%活性炭的20 mM磷酸溶液终止反应。未水解的GTP及蛋白将被活性炭吸附并通过离心沉降，随后通过闪烁计数检测分离出的无机磷酸的放射性活度。γ磷酸释放的初始速率及米氏动力学参数计算流程同上。 (2) T1343A突变型与野生型（WT）LRRK2质量光度分析原始数据分布图 质量光度（Mass Photometry, MP）实验参照已发表文献（Guaitoli等，2023）进行。实验采用Refeyn Two MP质量光度仪（Refeyn公司）检测LRRK2的二聚体比例。实验前，使用天然分子量标准品（Invitrogen，用含50 mM HEPES [pH 8.0]、150 mM NaCl及200 μM脱硫生物素的HEPES洗脱缓冲液按1:200稀释）建立颗粒对比度与分子量的标准曲线。质量光度检测前，将野生型或T1343A突变型LRRK2蛋白与0.5 mM ATP或缓冲液（对照组）在30 ℃孵育30 min。将LRRK2蛋白用洗脱缓冲液稀释至终浓度的2倍（终浓度分别为75 nM及100 nM），先在10 μL洗脱缓冲液中完成光学系统聚焦，再加入10 μL预处理后的蛋白样品。根据样品计数结果选择20 s至1 min不等的采集时长。每次测量的二聚体比例按公式进行归一化处理，实验设置三次生物学重复。 (3) 结合GDP/Mg或GTP/Mg的pT1343磷酸化LRRK2的AlphaFold3模型 本研究使用AlphaFold3（Abramson等，2024）构建并对比了磷酸化T1343位点的LRRK2在GDP结合态与GTP结合态的三维结构。有趣的是，AlphaFold3模型显示pT1343残基的磷酸基团向内取向，从而在LRRK2的GDP结合态中替代了GTP的γ磷酸基团。该发现与近期发表的分子动力学（Molecular Dynamics, MD）模拟结果高度一致（Stormer等，2023）。 (4) 基于细胞的磷酸化Rab检测实验的蛋白质免疫印迹（Western Blot）原始文件（对应预印本版本中图6补充材料2/补充图4的原始数据，Gilsbach等，2024） 基于细胞的LRRK2活性检测实验参照已发表文献（Singh等，2022）进行。简要流程：将HEK293T细胞培养于含10%胎牛血清及0.5%青霉素-链霉素的DMEM培养基中。实验时将细胞接种于六孔板，待细胞汇合度达50%~70%时，采用基于聚乙烯亚胺（PEI）的脂质转染法转染带有SF标签的LRRK2变体质粒。转染48 h后，用裂解缓冲液[30 mM Tris-HCl（pH7.4）、150 mM NaCl、1% NonidentP-40替代物、完整蛋白酶抑制剂混合物、PhosStop磷酸酶抑制剂（Roche）]裂解细胞。裂解液经10,000×g离心去除沉淀，调整蛋白浓度至1 μg/μL（使用1×Laemmli缓冲液）。随后将样品进行SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）及蛋白质免疫印迹分析，以检测LRRK2 pS935及Rab10 T73的磷酸化水平，具体流程如下：以总LRRK2及总Rab10蛋白水平作为归一化参照。免疫印迹实验采用NuPAGE 10% Bis-Tris凝胶（Invitrogen）进行SDS-PAGE分离，随后将蛋白转移至聚偏氟乙烯（PVDF）膜（Thermo Fisher）。为同时检测LRRK2与Rab10，将膜在140 kDa分子量Marker条带处水平切开。用含5%脱脂奶粉的TBST缓冲液（25 mM Tris，pH 7.4、150 mM NaCl、0.1% Tween-20）封闭非特异性结合位点（室温孵育1 h），随后在4 ℃下与一抗共同孵育过夜，一抗稀释比例如下：磷酸化特异性抗体用TBST/5%牛血清白蛋白（Roth GmbH）稀释，非磷酸化特异性抗体用TBST/5%脱脂奶粉（BioRad）稀释。磷酸化Rab10水平通过位点特异性兔单克隆抗体anti-pRAB10(pT73)（Abcam，ab230261）检测，LRRK2 pS935水平通过位点特异性兔单克隆抗体UDD2（Abcam，ab133450）检测，两者稀释比例均为1:2000。总LRRK2水平通过自研大鼠单克隆抗体anti-pan-LRRK2（克隆24D8；1:10000）（Carrion等，2017）检测。总Rab10水平通过兔单克隆抗体anti-RAB10/ERP13424（Abcam，ab181367）检测，稀释比例为1:5000。检测时使用HRP标记的山羊抗大鼠IgG或山羊抗兔IgG二抗（Jackson ImmunoResearch），稀释比例为1:15000（用TBST/5%脱脂奶粉配制）。抗原-抗体复合物采用ECL plus化学发光检测系统（GE Healthcare）显影，使用Stella成像系统（Raytest）进行成像及定量分析。 图6源数据1：展示了用于定量分析的Stella系统成像结果，注释文件对应图6补充材料2（Gilsbach等，eLife 2024，doi:10.7554/eLife.91083）。用于图6定量分析的条带对应LRRK2 pS935、总LRRK2、Rab10 pT73及总Rab10。