Limited proteolysis and interaction analysis via chromatography .1 Limited proteolysis .1 Limited proteolysis

GST-Atg16L1(328-623)

Murine GST-Atg16L1(328-623) was digested with proteases to characterize its stability and identify smaller, stable fragments for crystallization. Either sequencing grade trypsin dissolved in 1 mM HCl (Roche) or chymotrypsin (Roche) were added to 1 mg/mL purified GST-Atg16L1(328-623) diluted in 100 mM NaCl, 30 mM Hepes pH 7.5, 50 mM Tris-HCl pH 8.0 in a 1:200 (w/w) ratio. The digestions were incubated at RT. 40 µL

samples were taken before addition of protease and then at indicated time points after protease addition, combined with 3x loading buffer and immediately boiled at 95 °C for 3 min.

His-Atg5~Atg16L1(1-231)

Tryptic digestion was performed using the same ratio as above. Samples for the 15% SDS-PAGE gel were taken before addition of trypsin and after 45 min incubation at RT. The remaining digested protein was inactivated with 4 mM Pefabloc SC (Roche) a serine protease inhibitor.

His-Atg5~His-Atg16L1(1-265)~His-Rab33B(30-202)Q92L

The Atg5~Atg16L1(1-265) complex was pre-incubated with His-Rab33B(30-202)Q92L for 30 min at 4°C. The proteins were diluted to a final concentration of 1mg/mL in buffer comprised of 0.15 M NaCl, 30 mM HEPES pH 7.5. Trypsin, chymotrypsin, elastase, and proteinase K were added 1:200 (w/w) to the protein mixture.

Reactions containing chymotrypsin, elastase and proteinase K were adjusted to 50 mM Tris pH 8.0. The reaction proceeded at RT. Samples were taken after 5, 10, 15, 30, 60, and 120 minutes and combined with 5x Schägger Jagow loading buffer and boiled at 95

°C for 5 min for analysis on a Schägger Jagow gel.

2.7.2 Interaction analysis of cleavage products via chromatography His-Atg5~Atg16L1(1-231)

After proceeding as described for the complex in section 2.7.1, 1 M imidazole was then added to the reaction to make a final concentration of 20 mM to prevent nonspecific binding to the 1 mL HisTrap™ FF column (GE Healthcare). Washing and elution steps were done manually with a syringe. The column was equilibrated with 5 CV binding/wash buffer (200 mM NaCl, 50 mM Tris-HCl Buffer pH 8.0, 20 mM imidazole, pH 8.0). To maximize binding of the protein fragments to the column, the flow-through was reloaded onto the column four times. Next, the column was washed with 5 CV binding/wash buffer of which the first two wash fractions were comprised of 1.5 mL fractions and third and fourth of 1 mL fractions. 5 CV elution buffer (200 mM NaCl, 50 mM Tris-HCl pH 8.0, 500 mM imidazole, pH 8.0) were applied to the column and 1 mL fractions were collected.

His-Atg5~His-Atg16L1(1-265)~His-Rab33B(30-202)Q92L

The samples were digested with trypsin as described in section 2.7.1 for 60 min before the reaction was stopped with 4 mM Pefabloc. For anion and cation exchange chromatography, a Mono Q 5/50 GL (GE Healthcare) and a Mono S 5/50 GL (GE Healthcare) column were used. After equilibration with binding buffer (Mono Q=20 mM Tris pH 8.0; Mono S=20 mM MES pH 6.0), the digested sample was diluted to 10 mL with binding buffer and applied to the column. The column was washed with 5 CV (1 CV= 1mL) and the proteins were eluted with a gradient from 0-100% elution buffer (binding buffer containing 1 M NaCl) over 20 CV during which 1 mL fractions were collected.

His-Rab33B(30-202)Q92L~Atg16L1(141-265)

The samples were digested with trypsin as described above for 5 min before the reaction was stopped with 4 mM Pefabloc. For anion and cation exchange chromatography, a Mono Q 5/50 GL (GE Healthcare) and a Mono S 5/50 GL (GE Healthcare) column were used. After equilibration with binding buffer (Mono Q=20 mM Tris pH 8.0, 2 mM MgCl2; Mono S=20 mM MES pH 6.0, 2 mM MgCl2), the digested sample was diluted 1:4 with binding buffer and applied to the column. The column was washed with 5 CV (1 CV= 1mL) and the proteins were eluted with a gradient from 0-100% elution buffer (binding buffer containing 1 M NaCl) over 20 CV during which 1 mL fractions were collected.

His-Atg5~His-Atg16L1(1-106) ± D-myo-inositol-(1,3)-diphosphate [IP(1,3)2]

The proteases provided in the Proti-Ace™ (Hampton Research, HR2-429) and Proti-Ace™ 2 (HR2-432) Kits were first reconstituted in 100 µL double-distilled H2O.

The proteases were then diluted 1:10 in the dilution buffer provided in the kit. 5 µL of the previously diluted 0.1 mg/mL protease was pipetted into 5µL of the His-Atg5~His-Atg16L1(1-106) complex (10 mg/mL) either with or without 1 mM IP(1,3)2 that had been adjusted to 30 mM HEPES pH 7.5 and 0.2 M NaCl. The reaction was then incubated at RT or 37 °C , as indicated, and samples were taken after 5, 10, 15, 20, 30, and 60 minutes and combined with 2x Schägger Jagow loading buffer and boiled for 5 min at 95°C. For N-terminal sequencing, the procedure above was followed using clostripain and α-chymotrypsin as proteases. The reaction proceeded at RT for 5 and 10 min and the proteins were resolved on a Schägger Jagow gel.

2.7.3 Cleavage site determination via N-terminal protein sequencing

The protocol given by Seqlab GmbH (Goettingen, Germany) was followed. The sample was resolved by gel electrophoresis using an SDS-PAGE gel. The gel, Immobilon-FL Transfer PVDF membrane (Millipore), and 4 sheets of Whatman gel blotting filter paper (Whatman) were gently shaken in CAPS buffer. A semi-dry blotting apparatus (Phase, Lübeck) (Towbin 1979, 1989) was used: first two sheets of filter paper were placed on the bottom half of the apparatus. The gel was placed on top of this and the PVDF was placed on top of the gel. Two more sheets of filter paper completed the sandwich and bubbled were removed by gently rolling a pipet on top. The device was run at 45 to 50 mA for 20 to 45 minutes causing the negatively charged SDS-coated proteins to move upward toward the positive electrode and thus be transferred onto the membrane.

The blot was stained less than 1 min with the Coomassie Brilliant Blue because the acetic acid can interfere with sequencing. The membrane was allowed to dry overnight before the band was cut out and placed in a polypropylene cut for shipping.

2.8 Crystallization and structure determination