The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a “CPDCP-trunk” and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the “legumain-binding loop” of CY1 inhibit crayfish astacin following the “raised-elephant-trunk mechanism” recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and β only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.


Results and Discussion
Crystallization and proteolytic susceptibility of human fetuin-B in the presence of crayfish astacin. We have previously reported the structure of isolated mFB, which was recombinantly produced in mammalian cells and processed with endoglycosidase H 36 . This treatment clipped down the N-glycans attached to N 40 and N 139 (mFB amino-acid numbering in superscript and italics according to UniProt entry [UP] Q9QXC1) to single N-acetylglucosamine moieties. In particular, the sugar attached to N 40 participates in a packing contact that is crucial for crystal formation. In contrast, no crystals have been obtained with protein heterologously produced in insect cells, which attach different glycans. However, for the astacin·mFB complex crystal structure, protein from insect cells has been successfully employed in the presence of MP excess 36 . In this study, hFB was recombinantly produced and purified from mammalian cells, and crystallization of the astacin·hFB complex was also only successful with peptidase excess. SDS-PAGE, Western blotting, and N-terminal Edman degradation of the complex in solution revealed that most of the CTR was removed through cleavages at positions G 302 -S 303 (hFB residue numbering in superscript according to UP Q9UGM5), E 322 -A 323 and somewhere upstream within the CTR to yield species migrating according to ~30-35 kDa instead of the ~50 kDa of intact hFB (Fig. 1A,C). Cleaved forms were isolated by astacin affinity chromatography for comparative inhibitory studies (see Fig. 1E and below). In contrast to hFB, mFB proved more resistant to astacin degradation under equivalent conditions (Fig. 1B). This is consistent with the aforementioned crystallization experiments 36 , which revealed that peptidase excess cleaves mFB at bond S 296 -S 297 within the CTR of the otherwise intact molecule 36 . The structure of the mouse complex has shown that the CTR is an irregularly folded domain that is partially disordered and devoid of regular secondary structure, which explains its proteolytical susceptibility.
Astacin·hFB crystals diffracted to a similar resolution as those of the mouse complex (3.0 Å vs. 3.1 Å) and likewise contained two enzyme·inhibitor complexes per asymmetric unit (A·B and C·D). However, they belonged to a different space group (P6 1 vs. P2 1 2 1 2 1 ; compare Table 1 and Table 1 in 36 ), had different cell constants, and contained significantly more solvent (57% vs. 47%).
Structure of cleaved human fetuin-B. The structure spans domains CY1 (A 25 /N 27 -S 143 ) and CY2 (S 156 -E 258 /S 259 ), which are connected by the linking segment LNK (K 144 -P 155 ), plus a small fragment of the CTR (R 364 /T 365 -A 375 /S 376 ) ( Fig. 2A). Characteristic of cystatin-type domains, CY1 and CY2 consist of an antiparallel five-stranded curled β-sheet of up-and-down connectivity (strands β1-β5 in CY1 and β6-β10 in CY2) and a perpendicular α-helix (α1 in CY1 and α3 in CY2) inserted between the first two strands. The second/third and fourth/fifth strands of each sheet are joined by short connections (hairpins I and II, respectively), while the third and fourth strands are connected by long "legumain-binding loops" (LBLs) 36,43 of 21 and 19 residues in CY1 and CY2, respectively. In the latter domain, the LBL is disordered for C 216 -S 228 . In contrast, in CY1 it is well-defined and creates a hydrophobic pillow at its most exposed segment spanning I 108 -F 110 . Residing on this pillow, LNK comprises helix α2 and the CPDCP-trunk (C 151 -P 155 ), which protrudes from the molecular surface ( Fig. 2A). Finally, a 12-residue fragment of the CTR is attached to CY1 through disulfide C 36 -C 368 . Further disulfides are found in the LBLs (C 93 -C 104 in CY1 and, probably, C 216 -C 224 in CY2), as well as between β4 and β5 of CY1 (C 117 -C 137 ) and β9 and β10 of CY2 (C 237 -C 254 ). A last disulfide (C 151 -C 154 ) tightly crosslinks LNK to provide rigidity to the CPDCP-trunk. Within CY1, two N-glycan chains are attached to N 37 and N 136 , respectively ( Fig. 2A).
The two hFB moieties in the asymmetric unit (B and D) superpose with an rmsd of 1.1 Å. While the respective CY2 and LNK moieties fit well, the CY1 domains are rotated by ~5° around K 144 Cα, which leads to a displacement of maximally ~4.5 Å (at P 126 Cα) (Fig. 2B). In addition to this rigid-body displacement, which in general keeps the same conformation in both CY1 domains, it is remarkable that significant rearrangement is found in segment R 97 -M 106 within the LBL, which is displaced by ~5 Å maximally (at A 100 Cα).

Inhibition of crayfish astacin by human fetuin-B.
Crayfish astacin is a bipartite molecule of 202 residues consisting of two equally large upper and lower sub-domains (USD and LSD), which form an extended, deep active-site cleft at their interface 9,10,44 . The cleft harbors the catalytic zinc ion, which is bound by three histidines from a zinc-binding consensus sequence (H 92 -EXXHXXGXX-H 102 ; mature residue numbering of astacin in subscript; for numbering of the preproprotein according to UP P07584, add 49), which further contains the general base/acid for catalysis (E 93 ) and is a hallmark of the astacins 11,12 and other metzincin MPs 8,14,17 .
In the complex, the hFB moiety inserts like a chock into the active-site cleft of astacin through contacts made by the LNK, hairpins I and II of CY2, and the tip of the LBL of CY1 (Fig. 3A,B). This causes the cleft of both MP protomers in the asymmetric unit (A and C) to slightly open, triggered by a ~7°-rotation of the LSD around a horizontal axis traversing F 100 and P 176 , which causes a maximal displacement of ~3 Å (at S 123 Cα). Owing to the slight differences between hFB moieties B and D (see previous section), the A·B and C·D complexes also vary. Superposition of the respective MP moieties reveals that while the chock elements of the hFBs coincide, the cleft-distal parts of the inhibitors are rotated relative to each other by ~12° about a horizontal axis traversing T 150 and V 201 , which causes a displacement of ~11 Å maximally (at P 126 ; see Fig. 3C). This is reminiscent of the complex between the otherwise unrelated MP thermolysin from Bacillus thermoproteolyticus and its specific inhibitor IMPI from the greater wax moth Galleria mellonella 45 . The latter likewise follows a wedge-like inhibition mechanism involving a few structural elements on the inhibitor surface. In the two complexes in the asymmetric unit, the inhibitor moieties are rotated by ~5° relative to each other by visual inspection.
In the hFB·astacin complex A·B (Fig. 3A,B), the interface spans 1,245 Å 2 , with a theoretical solvation energy gain on complex formation (Δ i G according to 46 ) of −20.1 kcal/mol and a significant complexation significance

Dataset
Crayfish Astacin·Human Fetuin-B www.nature.com/scientificreports www.nature.com/scientificreports/ score (CSS according to 46 ) of 0.50. The interface involves 35 residues of hFB and 41 of astacin, which make 14 hydrogen bonds, two ionic interactions, and hydrophobic interactions between 27 pairs of residues (see Table 2). Astacin segments involved in the complex are S 62 -V 68 , Q 80 -H 102 , S 126 -Y 133 , and Y 149 -W 158 . Participating hFB regions include I 108 -E 111 from the LBL of CY1 and Y 148 -P 155 from helix α2 plus the downstream CPDCP-trunk, which from M 149 to D 153 runs along the non-primed side of the cleft in the direction and orientation of a substrate (Fig. 3B). In particular, D 153 binds the catalytic zinc of astacin according to an aspartate-switch mechanism, as well as the general base/acid E 93 and Y 149 . After D 153 , the polypeptide performs a ~90°-kink outward and leaves the cleft, which explains why fetuin-B is not cleaved at bond D 153 -C 154 . This contrasts with standard-mechanism inhibitors 47 of MPs, such as the aforementioned IMPI from which a reactive-site loop is inserted like a substrate across the entire cleft of thermolysin. Thus, the reactive-site bond that links residues in sub-site positions S 1 and S 1 ' (for peptidase active-site cleft sub-site nomenclature, see 48,49 ) is cleaved 45 .
Assisting the CY1 LBL and the CPDCP-trunk, the tip of hairpin I of CY2 spanning Q 198 -F 206 blocks sub-sites on the primed side of the cleft (Fig. 3B). This hairpin contains segment Q 198 -WVVG-P 203 , which conforms to the hallmark sequence motif of inhibitory fetuins (QWVXGP 36 ), and V 200 and V 201 nestle into sub-sites S 2 ' and S 3 ', respectively. Finally, the tip of CY2 hairpin II (S 240 -F 249 ) further contributes to the complex by blocking outmost cleft sub-sites and attaching to the right surface of the enzyme (Fig. 3B). Overall, these interactions conform to the raised-elephant-trunk mechanism of astacin inhibition previously described in detail for mFB 36 . Thus, the present structure suggests that the excised CTR is not required for inhibition.   Fig. 2A, astacin is in salmon, and the catalytic zinc ion is shown as a magenta sphere for reference. The orientation results from that of Fig. 2A after a horizontal downward rotation of ~40°. This corresponds to a ~90°-rotation downward with respect to the standard orientation of astacin (see 49 ). (B) Close-up view of (A) depicting the principal residues engaged in complex formation of astacin (pink ribbon and carbons; residue numbers in dark red) and hFB (tip of CY1 LBL as an orange ribbon, LNK as a red ribbon, CY2 hairpin I as a turquoise ribbon, and CY2 hairpin II as a green ribbon) with carbons in pale blue (residue numbers in blue). (C) Superposition of the enzyme moieties of the two astacin·hFB complexes A·B (astacin in sky blue, CY1 in yellow, LNK in red, CY2 in white, and CTR in pink) and C·D (astacin in blue, CY1 in orange, LNK in green, CY2 in gray, and CTR in purple) reveals flexibility of domain CY1. www.nature.com/scientificreports www.nature.com/scientificreports/ As expected from a comparison of the isolated inhibitor moieties (see previous section), superposition of the peptidases of the two complexes in the asymmetric unit reveals that the segments engaged in inhibition (tip of CY1 LBL, LNK with CPDCP-trunk and hairpins I and II of CY2), as well as the rest of CY2 (Fig. 3C), appear well-aligned. Hence, the aforementioned interactions at the A·B enzyme·inhibitor interface are also found in the C·D complex with just a couple of exceptions (Table 2). In contrast, the rest of CY1 with the attached CTR fragment significantly deviates in both complexes owing to a ~12°-rotation about K 144 , which causes a displacement of ~11 Å maximally, at the cleft-distal edge around G 77 Cα and S 127 Cα (Fig. 3C). In line with these differences, in complex C·D the interface spans 1,421 Å 2 , with a Δ i G of −20.4 kcal/mol and a CSS of 0.43. A total of 40 residues of hFB and 43 of astacin participate in the interface through 13 hydrogen bonds, two ionic interactions and hydrophobic interactions between 26 pairs of residues (see Table 2). Taken together, these findings support an ancillary role for CY1 in inhibition and, possibly, a tethering role for the missing CTR to fix the relative orientation of CY1 and CY2, although crystal packing may also play a role.
inhibitory studies in vitro. To assess the role of the CTR and the arrangement between CY1 and CY2 in peptidase inhibition, we first determined the apparent inhibition constant K i of intact hFB in front of crayfish astacin and human meprin α and β as a control of the generally valid inhibition of astacins by hFB, and found values in the subnanomolar range (Fig. 1D). These values were indicative of very potent inhibition and compared well with those of mFB (see Fig. 3 in 36 ). Next, we isolated astacin-cleaved hFB (see above), which entailed treatment with urea to dissociate the inhibitor from the peptidase, and determined its K i values in front of the aforementioned enzymes. As a control, we also determined these parameters for intact hFB subjected to the same chaotropic treatment (Fig. 1F). We found that urea slightly decreased the inhibitory power of intact hFB. However, the cleaved inhibitor still evinced potent inhibition with K i values in the nanomolar range (Fig. 1D,F). We conclude that the CTR and the relative orientation of CY1 and CY2 play a negligible role in the inhibition of astacin MPs.
Comparison with the mouse fetuin-B·crayfish astacin complex. Despite the lack of almost the entire CTR in the human inhibitor, but consistently with a sequence identity of 67% based on the CY1-LNK-CY2 domains involved in enzyme·inhibitor contact (Fig. 4A), the general inhibition modes of CTR-nicked mFB and CTR-depleted hFB are very similar and generally encompass the same segments, which have been outlined above and extensively described in 36 . However, significant differences can be found on detailed comparison.
First, in contrast with hFB, the two astacin·mFB complexes within the asymmetric unit evince only minor variations, mainly in the CTRs, as reflected by an rmsd of 0.85 Å. In the mouse complex, the complex interface spans 1,059 Å 2 and gives a Δ i G of −10.8 kcal/mol and a CSS of 0.33. The interface involves 33 residues of fetuin-B and 38 of astacin, which perform ten hydrogen bonds, two ionic interactions and 20 hydrophobic interactions 36 . All these values are significantly lower than those for the two astacin·hFB interfaces (see above and  Table 2. Interactions at the interface between astacin and human fetuin-B. The first residue/atom belongs to fetuin-B, the second to astacin. The two distances indicated for electrostatic interactions correspond to the A·B and C·D complexes, respectively. Hydrophobic interactions are for both the A·B and C·D complexes, if not otherwise stated in parenthesis. pointing to looser interaction in the mouse complex. This difference in inhibitory action is apparently reflected by the disparate serum concentrations of mFB (156 ± 3 µg/ml) and hFB (5 ± 1 µg/ml) 50 . Second, superposition of the entire complexes reveals rmsd values of 1.9 Å and 2.2 Å for mFB compared with hFB complexes A·B and C·D, respectively. If the respective peptidase moieties are superposed, the leftmost part of the chock comprising LNK and the LBL tip of CY1 (I 108 -F 110 in hFB and M 111 -Y 113 in mFB) neatly coincide for their main chains and most interactions are equivalent, including those of the CPDCP-trunk aspartate (D 153 in hFB and D 156 in mFB) (Fig. 4B). However, the rest of the fetuin-B moieties diverge substantially. Within the coil connecting α3 and β7 of CY2, deviations of ~10 Å and ~8 Å are detected between mFB K 186 Cα and topologically equivalent K 185 Cα from hFB molecules B and D, respectively. Similarly, mFB R 130 Cα and equivalent S 127 Cα at the tip of CY1 hairpin II are ~7 Å apart in both hFB molecules B and D. Even within the better fitting parts of the complexes, the first two residues of the second turn of LNK helix α2 of hFB (Y 148 -M 149 ) penetrate the non-primed side of the cleft of astacin more deeply than the equivalent pair of mFB, H 151 -T 152 (Fig. 4B).
Third, both hairpins I and II of CY2 on the primed side of the cleft significantly diverge. While the former has the same length in both fetuins and likewise comprises the hallmark motif QWVXGP (Q 199 -WVSG-P 204 in mFB), in hFB it intrudes the astacin cleft more deeply owing to a relative ~90°-rotation of the Cα-C bond of the tryptophan. This causes the side chain of the downstream valine to enter the S 1 ' sub-site while in mFB it merely performs a hydrophobic interaction with disulfide C 64 -C 84 on the top of the active-site cleft (Fig. 4B). In addition, hairpin II is eight residues shorter in hFB and, while slightly flexible at its tip (H 245 -R 246 ), it is clearly resolved in the final Fourier map. In contrast, it is disordered and untraceable for four and six residues in the two mFB complexes. Thus, interactions of hFB with Y 177 -D 178 and W 158 of astacin that could not be detected in the mouse complex were defined here ( Fig. 4B and Table 2). Interestingly, mFB stands alone with its long CY2 hairpin II, which uniquely contains seven-to-nine residues more than fetuin-Bs from other animals, including the evolutionarily closely related naked mole rat (see Suppl. Fig. 3 in 36 ). This suggests that the other orthologs will probably have a structured CY2 hairpin II and bind astacins more similarly to hFB than to mFB.

conclusions
We crystallized the complex between hFB and crayfish astacin in the presence of an excess of the MP, which proteolytically removed most of the CTR but retained the complex-formation and inhibitory capacities of the fetuin. As found for the mouse ortholog, hFB blocked astacin following a raised-elephant-trunk mechanism by inserting like a chock into the active-site cleft but sparing sub-sites S 1 and S 1 '. This explains why hFB was not cleaved at the inhibitory loops in contrast with standard-mechanism inhibitors.
The two astacin·hFB complexes in the asymmetric unit evinced significant differences in the relative arrangement of CY1 and CY2. This differs from the mouse complex, which was only nicked at the CTR upon incubation with an equivalent excess of astacin and for which the two complexes in the asymmetric unit were similar. This indicates that the proteolytic susceptibility of the human and mouse fetuin-B orthologs vary despite close relatedness based on high sequence identity. In the mouse complex, the interaction surface and the theoretical solvation energy gain on complex formation were significantly smaller than in the human CTR-depleted complex, which points to a looser enzyme·inhibitor interaction of the intact mouse inhibitor.
Biochemical assays in vitro revealed that intact hFB and mFB inhibited crayfish astacin and human meprin α and β with comparable potency, with K i values in the subnanomolar range. This suggests similar inhibition mechanisms for both full-length orthologs and indicates that the unstructured CTR has a function in structural maintenance of a particular relative disposition between CY1 and CY2 in fetuins. This fixed arrangement probably limits the adaptive capacity of the intact inhibitor to produce an optimal interaction surface with the target MP as large as that of the cleaved form. Notably, the K i values of cleaved hFB in front of crayfish astacin and meprins α and β were still in the nanomolar range.

Materials and Methods
Protein production and purification. Protein hFB with a C-terminal hexahistidine-tag, thus spanning residues C 16 -P 382 -IEGRHHHHHH, was expressed and secreted to the extracellular medium by mammalian ExpiCHO-S cells according to the manufacturer's instructions (ThermoFisher Scientific) as previously reported 35 . Human meprin β was obtained as a zymogen as described 51 and activated with trypsin 52 . Protein mFB with a C-terminal hexahistidine-tag was cloned in vector pFASTBac1 and expressed in baculovirus-transduced High Five cells as described for meprin 35,51,53 . Human meprin α was produced as described 53 . All proteins used for crystallization were purified by nickel-nitrilotriacetic-affinity and size-exclusion chromatography steps as published 33,35 . Astacin was purified from the digestive fluid of the European freshwater crayfish Astacus astacus as reported 10 . www.nature.com/scientificreports www.nature.com/scientificreports/ (Catalonia, Spain) and kept at 100 K in the cryostream of an Oxford Cryosystems 700 series. Diffraction data was collected on a Pilatus 6 M pixel detector (Dectris) and processed with programs XDS 55 and XSCALE 56 , and converted to the MTZ-format used by the CCP4 suite of programs 57 with XDSCONV. Crystals belonged to space group P6 1 , contained two peptidase·inhibitor complexes per asymmetric unit (molecules A·B and C·D, respectively), and diffracted to 3.0 Å resolution (see Table 1 for data processing statistics).
Structure solution and refinement. The structure of the astacin·hFB complex was solved by maximum-likelihood-scored molecular replacement with the PHASER program 58 . Initial searches with the entire astacin·mFB complex (PDB 6HT9 36 ) were unsuccessful. Subsequent searches with the separate astacin and mFB moieties yielded chemically reasonable solutions upon visual inspection with a log-likelihood gain of 2,714 at final Eulerian angles and fractional cell coordinates (α, β, γ, x, y, z) 8. 162. The crystal packing was acceptable, and the log-likelihood gain was 2,508. Thereafter, automated model building was used to input the sequence of hFB and density modification was carried out with twofold averaging with the AUTOBUILD routine of program suite PHENIX 59 , which resulted in a Fourier map that was used for manual model building with the COOT program 60 . The latter alternated with crystallographic refinement with PHENIX 61 and BUSTER/TNT 62 under inclusion of non-crystallographic symmetry restraints and translation/ libration/screw-rotation refinement until completion of the model. The final refined model comprised residues A 1 -S 199 and A 1 -L 200 of astacin molecules A and C, respectively, plus a zinc cation each; segments A 25 -P 215 + S 229 -E 258 + R 364 -S 376 and N 27 -P 215 + S 229 -S 259 + T 365 -A 375 of hFB molecules B and D, respectively; and 31 solvent molecules (see Table 1 for the final model statistics). Residues N 37 of molecules B and D had the glycan structure NAG(FUC)-NAG-BMA attached (for sugar moiety nomenclature, see the legend of Table 1). Residues N 136 of molecules B and D showed only two and one NAG moieties attached, respectively. The quality of the final model was assessed with the wwPDB X-ray Structure Validation Service (https://validate.wwpdb.org 63 ). proteolytic susceptibility of human and mouse fetuin-B. Both inhibitors were incubated with astacin in 150 mM sodium chloride, 50 mM Tris·HCl, pH 7.4. N-terminal sequencing by Edman degradation was performed by Proteome Factory AG (Berlin, Germany). Immunoblot analysis was performed as reported 35 using a polyclonal hFB antibody as reported 50 and a penta-His-antibody (Qiagen, Hilden, Germany). Cleaved hFB was isolated from the complex by astacin affinity chromatography. To this aim, astacin was covalently coupled to a HiTrap NHS-Activated HP affinity column (GE Life Sciences, Freiburg, Germany) according to the manufacturer's specifications. Protein hFB was applied to the column and cleaved by addition of further astacin. The cleaved hFB was eluted by addition of 6 M urea, desalted and concentrated. The non-cleaved hFB control was treated similarly except for cleavage by astacin. The residual inhibitory capacities of both samples against astacin and human meprins α and β were determined. inhibition assays in vitro. Inhibition of meprin α, meprin β and astacin by intact and astacin-cleaved hFB was determined in vitro in a Varioskan Flash 3001 spectral plate reader with SKANIT 2.4.3.RE software (ThermoFisher Scientific, Dreieich, Germany) with a fluorescence-based activity assay as previously described for mFB 36 . Enzyme concentrations were determined by absorbance at 280 nm (ε astacin = 42,800 M −1 cm −1 ; ε meprin α = 106,520 M −1 cm −1 ; ε meprin β = 113,385 M −1 cm −1 ) and assays were performed in triplicate at 37 °C in 100 µl final volume, with 150 mM sodium chloride, 50 mM Tris·HCl, pH 7.4 and 0.01% Brij-35 as buffer. Cleavage reactions were initiated by the addition of 20-30 µM Ac-R-E(Edans)-D-R-Nle-V-G-D-D-P-Y-K(Dabcyl)-NH 2 (Biosyntan, Berlin, Germany) for meprins α and β, and 170-180 µM Dansyl-P-K-R-P-W-V-OH (PANATecs, Heilbronn, Germany) for astacin, both predissolved in dimethyl sulfoxide (final concentration 0.4%). Initial velocities were recorded for at least 600 s (100 times for 100 ms at intervals of 15 s). Thereafter, 1.5 µl of proteinase K (Sigma-Aldrich) at 20 mg/ml or 1 µl astacin at 100 µM were added for complete substrate turnover. The latter was monitored and subsequently calculated using the formula v = [S] × m/ΔF, where [S] is the substrate concentration, m the [F/t] slope of initial linear substrate turnover, and ∆F the maximal fluorescence intensity corresponding to complete turnover. Kinetic parameters of inhibition (K i ) were determined using Morrison's equation 64 . Bioinformatics. Structure figures were prepared with the CHIMERA program 65 . Structure superimpositions were performed with SSM 66 within COOT. Protein interfaces were analyzed with PISA 46 (www.ebi.ac.uk/pdbe/pisa). The area of the interface of a complex was taken as half the sum of the buried surface areas of either molecule. Sequence identities were calculated with SIM with default parameters (https://web.expasy.org/cgi-bin/sim/sim. pl?prot). Signal peptides were predicted with SIGNALP v. 5.0 at http://www.cbs.dtu.dk/services/SignalP-5.0 67 . The final coordinates of the crayfish astacin·human fetuin-B complex are available from the PDB at www.rcsb.org (code 6SAZ).