Supplemental Experimental Procedures Cloning, Protein Expression and Purification

identified by expressing different constructs of the protein and subjecting them to limited proteolysis using various proteases (e.g. trypsin, chymotrypsin, GluC, AspN, thermolysin). Stable fragments were identified by N-terminal sequencing and mass spectrometry. The fragment N39-K364 was cloned into the GATEWAY pDEST17 expression vector (Invitrogen). The protein has an N-terminal tag consisting of six histidines which was cleaved off with TEV protease after purification. All point mutations (T208A/S212A, K82R) were subcloned from existing plasmids (Timm et al., 2003). Proteins (wild type and mutant) were expressed in E.coli strain BL21 AI (Invitrogen). Cells were induced overnight at 24°C by adding 0.2 % arabinose at OD600 ≈ 0.6. Cells harvested from a 1.5 L culture were resuspended in 40 ml lysis buffer (50mM Hepes pH 7.2, 300 mM NaCl, 5 % glycerol supplemented with one tablet EDTA-free complete protease inhibitor cocktail (Roche)) and lysates were prepared by passing twice through a French press cell. The expressed proteins were purified in four steps: Ni-NTA affinity chromatography, TEV protease cleavage to remove the His-tag, ion exchange chromatography and gel filtration. Clarified lysates were applied to a Ni-NTA affinity column of 5ml Ni-NTA beads (Qiagen