Supplementary Figure 1 Preparation, crystallization and structure determination of EpEX. (a), Purified EpEX and EpEX analyzed on homogenous 12.

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1 Supplementary Figure 1 Preparation, crystallization and structure determination of EpEX. (a), Purified EpEX and EpEX analyzed on homogenous 12.5 % SDS-PAGE gel under reducing and non-reducing conditions. Single sharp band corresponding to EpEX indicates higher sample homogeneity that of EpEX. Here, several closely spaced bands, all at the higher M r than EpEX, indicate heterogeneous glycosylation. (b), EpEX crystal. (c), Anomalous Fourier map shows the position of sulfur atoms as identified by S-SAD phasing. EpEX polypeptide chain is shown as tube where width and coloring corresponds to B-factors (thin and blue - low B-factor, wide and red high B-factor) associated with poorly defined electron density. All 16 intrinsic sulfur atoms of 12 cysteine and 4 methionine residues (red sticks) are shown as yellow balls. The map (grey mesh) is contoured at 4σ level. Positions of 14 of the total 16 sulfur atoms were determined using this map. No anomalous signal was observed for sulfur atoms of disordered side chains of Met231 and Met261. Some parts of the polypeptide chain of EpEX do not have a well-defined electron density and are associated with high B-factor values: Val51 to Gln54 (loop between βa2 and βa3 strands within ND), Glu132 to Val139 (last part of TY domain), Ser199 to Glu206 (region between βc3 strand and α3 helix) and Lys229 to Asp232 (central part of the RCD region). These segments mostly correspond to parts of polypeptide chain which are exposed to the solvent in the EpEX dimer.

2 Supplementary Figure 2 Crystallization additive n-decyl-β-d-maltopyranoside (DMU) is bound in a narrow hydrophobic pocket in EpEX. (a) Structure of n-decyl-β-d-maltopyranoside. (b) Ribbon representation of EpEX cis-dimer color-coded as in Fig. 2a. The two DMU molecules (one per each subunit) are shown as sticks (carbon atoms in grey, oxygen atoms in red) with transparent surface. (c) DMU fitted in a long electron density blob. DMU is shown as sticks (carbon atoms in grey, oxygen atoms in red). The 2F o - F c electron density map (grey mesh) is contoured at 1.3σ level. The inside of the pocket in which the detergent s tail is bound is lined by hydrophobic side chains of several amino acid residues of α2 and α3 helices plus βc sheet. The polar head of DMU interacts with polar side chains of residues located at the mouth of the pocket including the RCD. (d) EpEX cis-dimer shown as molecular surface colored according to electrostatic potential and sliced to reveal the hydrophobic pocket with bound DMU (shown as sticks). Also visible is the polar head of DMU bound to the other subunit. Position of α2 (view along the helix axis) and α3 helices (view perpendicular to the helix axis) is denoted by circle and rectangle, respectively. In this view α3 helix is behind the pocket.

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4 (continued) Supplementary Figure 3 Evolutionary conservation of EpEX. (a) Alignment of EpEX sequences from species belonging to several different groups (PRIM primates, UNGU ungulates, ARMA armadillos, CARN carnivores, WHDO whales and dolphins, RODE rodents, BATS bats, PIKA pikas, MARS marsupials, BIRD birds, REPT reptilians, AMPH amphibians, FISH fishes). Numbering of amino acid residues, secondary structure elements, and RCD and TY-loop regions correspond to human EpEX. The three N-glycosylation sites of wt human EpEX are marked with red hexagons. (b) Sequence identity as inferred from alignments of EpEX sequences in a (excluding birds, reptilians, amphibians and fishes), mapped to structure EpEX cis-dimer which was back-mutated in silico to include all three N-glycoslyation sites. The pyroglu24 was changed to Gln. The ribbon (above) and surface representations (below), both depicting EpEX cis-dimer in three different orientations relative to the membrane (gray bar), are gradient-colored according to sequence conservation with the two extremes at blue (more conserved) and red (less conserved). For clarity, the C-terminal region of EpEX (from Met261 to Lys265 plus His266) was removed. Different EpEX surface regions are outlined (dotted line).

5 Supplementary Figure 4 Close-up of membrane proximal EpEX region reveals the C-terminal part in unnatural orientation. EpEX cis-dimer is shown in cartoon representation color-coded as in Fig. 1. TY-loop is colored yellow. C-terminal residues (Glu258-His266) and residues within 5 Å radius are shown as sticks, water molecules are depicted as red spheres. 2F o - F c electron density map (grey mesh) of the Met216-His266 region is contoured at 1.2σ. Only the first histidine residue (His266) of the His 6 -tag has a defined electron density. O H266 denotes carbonyl oxygen atom of His266.

6 Supplementary Figure 5 EpEX and glycosylated EpEX are cleaved by cysteine cathepsins within the TY-loop. (a, b) Electrophoretically separated fragments under reducing conditions of (a) EpEX (wt, i.e. glycosylated) and (b) EpEX generated by incubation of intact protein samples with cathepsins L, S, K or B. Two different molar ratios of cathepsin over EpEX( ) were used (10-3 and 10-2 ). Each pair of EpEX( )-cathepsin was incubated at slightly acidic ph (optimum ph for cathepsin activity) and at ph 7.2. Most potent in generating the cleaved form of EpEX( ) are cathepsins L and K whereas cathepsins S and B cleave EpEX( ) only at low ph and to a small extent. Primary cleavage site for cathepsins L and K is Gly79-Arg80 with an additional minor cleavage site Leu78-Gly79 for cathepsin L.