Protein preparation

For crystallization, N-Cbl Y371F (residue 47–435) was expressed, purified and stored as described previously [6]. For SPR and SAXS analysis, N-Cbl variants were cloned into a modified form of pGEX4T1 (GE Healthcare) containing an N-terminal histidine glutathione S-transferase (His-GST) tag followed by a thrombin or TEV-protease cleavage site and expressed in E. coli BL21 (DE3) Gold. pTyr371-N-Cbl was generated as described previously [6]. For SPR analysis, all N-Cbl variants were purified by Ni-NTA followed by glutathione-affinity chromatography. Anion exchange chromatography was subsequently used to separate His-GST-pTyr371-N-Cbl from unphosphorylated His-GST-N-Cbl. For SAXS studies, N-Cbl variants were then treated with TEV or thrombin protease to cleave the His-GST tag and further purified by Ni-NTA pass-back followed by anion exchange and size exclusion chromatography. Proteins for SAXS were stored in 25 mM Tris-HCl (pH 7.6), 500 mM NaCl and 1 mM DTT at –80 °C. UbcH5B S22R C85K–Ub (referred to as UbcH5B–Ub) was expressed, generated and purified as described previously [7]. His-GST-tagged protein concentrations were determined by Bradford assay using BSA as a standard and all other concentrations were determined using a NanoVue Spectrophotometer (GE Healthcare).