Protein production and purification.

For protein production E. coli BL21(DE3) (New England Biolabs, Frankfurt, Germany) cells carrying the pET24C overexpression plasmids were grown in LB medium, supplemented with kanamycin (50 μg ml⁻¹), to an OD₆₀₀ of 0.3. After induction with IPTG (0.1 mM) or d-(+)-lactose-monohydrate (12.5 g liter⁻¹), the cultures were incubated overnight at 15°C under rigorous shaking. After cells were harvested by centrifugation (5,000 rpm, 20 min, 4°C), the pellet was resuspended in HEPES buffer (20 mM HEPES, pH 8.0, 250 mM NaCl, 20 mM MgCl₂, and 20 mM KCl) for pulldown experiments or in Tris-HCl buffer (50 mM Tris-HCl, 250 mM NaCl, 25 mM KCl, 5 mM MgCl₂, 0.5 mM dithiothreitol [DTT], 0.01% sodium azide, 5% glycerol, pH 7.5) for degradation assay, supplemented with 40 mM imidazole (buffer A). Subsequently, the cell suspension was lysed by sonification (Bandelin Sonoplus), cell debris was removed by centrifugation (20,000 rpm, 30 min, 4°C), and the supernatant was filtered and loaded on a 1-ml or 5-ml HisTrap column (GE Healthcare), which had previously been equilibrated with 5 column volumes (CV) of buffer A. After cells were washed with 5 CV of buffer A, the protein was eluted with 5 CV of buffer B (the corresponding protein buffers supplemented with 300 mM imidazole). The protein was then concentrated using an Amicon Ultracel‐10K (Millipore). The concentrated sample was applied to size exclusion chromatography (SEC) (HiLoad 26/600 Superdex, 200 pg; GE Healthcare) equilibrated with the corresponding protein buffers. Protein concentration was determined by a spectrophotometer (NanoDrop Lite, Thermo Scientific). For storage, the glycerol concentration was elevated to 20%, and the protein was snap-frozen in liquid nitrogen and stored at 20°C.