Gppnhp

GppNHp-bound RAS isoforms were generated by exchanging GDP-bound versions purified above with non-hydrolysable GTP analogues. In brief, EDTA (25 mM final) and 24-fold molar excess GppNHp (Jena Biosciences, 100 mM solution in 0.1 M Tris pH 8) were added to 0.5–1 μmol of purified RAS to obtain a final volume of 2.5 ml, and incubated 1 h at room temperature. PD-10 size-exclusion gravity columns (Cytiva) were equilibrated with nucleotide-exchange buffer (40 mM Tris pH 8, 200 mM (NH₄)₂SO₄, 0.1 mM ZnCl₂) according to the manufacturer’s instructions. The RAS sample was loaded onto the column and eluted with 3.5 ml of nucleotide exchange buffer. A fresh 24-fold excess of GppNHp was added, along with 50 μl of shrimp alkaline phosphatase (rSAP, NEB, M0371S). The solution was incubated for 1 h at 4 °C. To quench, 210 μl of 1 M MgCl₂ was added to a final concentration of 30 mM. The solution was then diluted with SEC buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 10 μM GppNHp, 5 mM MgCl₂) to about 10 ml, before final purification on a Superdex 75 HiLoad 26/600 size-exclusion column. Pure fractions were pooled, concentrated and frozen at 10–20 mg ml⁻¹.

For the pulldown with full-length RILPL2, nucleotide exchange was performed using purified WT Rab8a incubated in 10 mM EDTA for 10 minutes at room temperature in the presence of 10X molar excess GDP. The exchange was terminated by addition of 15 mM MgCl₂ and excess nucleotides were removed by running samples through a PD10 column (GE healthcare), or by immediate gel filtration chromatography. To verify successful exchange, 100 μL the protein (>1mg/mL) was boiled for 10 min at 95°C to denature the protein and release the nucleotide, followed by centrifugation for 30 min 16,000 x g, 4°C to remove precipitated protein. The supernatant was mixed with running buffer (100 mM potassium phosphate, 8 mM thiobarbituric acid, pH 6.5) at a 1:1 ratio. The samples were loaded on an Acquity Ultra Performance system (Waters Corporation, Milford, MA, USA; or Varian 920 LC machine, Agilent, Stockport, UK) equipped with a ZORBAX 300SB-C18 column (Agilent, Stockport, UK). Elution profiles of GMP, GDP, GTP (Sigma Aldrich) and GppNHp (Jena Bioscience, Germany) were subjected to HPLC and compared with Rab8a. The nucleotide state of Rab8a(Q67L) was confirmed to be GTP-bound using the analytical HPLC strategy.

MST (NanoTemper Technologies GmbH) was employed to determine the mutation effect on the binding affinities of RAB33B and ATG16L1. RAB33B proteins were labeled with Tide Fluor 3 (TF3) maleimide (AAT Bioquest, 2270). Labeled RAB33B was used at a concentration of 10 μM. Unlabeled ATG16L1(141–265) was added in a 1:1 dilution in a concentration from 400 μM to 12 nM. Samples were prepared in a buffer containing 20 mM HEPES (Carl Roth, 6763.3), pH 7.2, 25 mM NaCl (Carl Roth, P029.2), 2 mM DTE (Sigma, D8255), 2 mM MgCl₂ (Carl Roth, HN03.1), 0.05% Tween 20 (Sigma, P1379) and 10 µM GDP (Sigma, G7127) or GppNHp (Jena Bioscience, NU-899). The samples were filled into standard capillaries and measured with a 38% LED and 60% IR-Laser with laser-on time of 30 s and laser-off time of 5 s. The movement of the molecules along the temperature gradient results in a reduction of fluorescence. Plotting the reduction of fluorescence against the concentration of the titrant ATG16L1(141–265) yielded a non-linear binding curve. The binding curves were fitted with the nonlinear solution of the law of mass action to obtain the dissociation constant.

RAB33B proteins with a defined nucleotide-binding state were obtained by nucleotide exchange. Ethylenediaminetetraacetic acid (EDTA) (Sigma, E9884) at a final concentration of 5 mM (2.5-fold higher than the concentration of MgCl₂) was added into the RAB33B protein. Subsequently, 20-fold excess of GppNHp (Jena Bioscience, NU-899), GTP or GDP was added and the solution was incubated at room temperature for 2 h. The protein solution was subjected to buffer exchange [20 mM HEPES (Carl Roth, 6763.3), pH 7.2, 25 mM NaCl (Carl Roth, P029.2), 2 mM DTE (Sigma, D8255), 2 mM MgCl₂ (Carl Roth, HN03.1) and 10 µM respective nucleotide)] using a NAP5 column (GE Healthcare, 17–0853-01) to remove excess EDTA (Sigma, E9884) and nucleotide. The state of the nucleotide binding was determined by high-performance liquid chromatography using ProntoSIL 120-5-C18-AQ 5 µm (250 x 4.6 mm) column (Bischoff) eluted with buffer [50 mM potassium phosphate (prepared from dipotassium hydrogen phosphate, Sigma, P3786), pH 6.6, 10 mM tetrabutylammonium bromide (Sigma, 193119), 8% acetonitrile (Sigma, 34851)] in a Waters LC system (Waters Corp.).

Full-length DrrA was amplified from L. pneumophila genomic DNA in previous work^{13 (link)}. For E. coli expression, DrrA truncation constructs were cloned into a modified pSF vector (a gift from Stefanie Pöggeler lab, Georg-August-University Göttingen) via Gibson assembly using Gibson Assembly Master Mix (New England Biolabs, Frankfurt, Germany). Constructs for Rab1 and Rab8a were reported previously^{13 (link),45 (link)}. All point mutations were performed with a Q5 Site-Directed Mutagenesis Kit (New England Biolabs). All plasmids were confirmed by DNA sequencing. Plasmids for genetic code expansion experiments were designed and cloned as described in the Supplementary Information. The synthesis of BrC6K has been reported in detail in Cigler et al.^{19 (link)}. GppNHp and GDP were purchased from Jena Bioscience, Jena, Germany. The synthesis of TReNDs has been reported previously^{24 (link)}. Tobacco etch virus (TEV) protease was produced in-house. Lipofectamine LTX for transient transfection in H1299 cells was purchased from Thermo Fisher Scientific (Darmstadt, Germany). MTS agent was purchased from Promega, Walldorf, Germany. Unless otherwise stated, all other reagents were purchased from Sigma (Taufkirchen, Germany) and Carl Roth (Karlsruhe, Germany).