Fig. 1. Schematic representation of the domain-specific structure of otolin-1 based on UniProt data for otolin-1 with identifiers A5PN28 and A6NHN0 (A). Given segments (SP ? signal peptide; NC-N? ? noncollagenous N-terminal domain; central collagen-like domain and C-terminal globular C1q domain) are captioned with amino acid positions in the full primary sequence of the protein. Panel B shows fragments of otolin-1 trimeric structure models composed of the collagen-like domain 3 and C1q domains.

Fig. 2. Circular dichroism spectra of dOtol1 and hOtol1 in far (A) and near (B) ultraviolet light in buffer F. For far-UV CD spectroscopy, a 10-?M protein concentration was used, while for near-UV CD, the protein concentration was 40 ?M. The internal column graph in A shows the structure content after spectral deconvolution with CDNN 2.1. software. The near-UV CD spectra regions (B) are marked in grey corresponding to signals derived from Trp, Tyr and Phe residues in the range of 285?305 nm, 275?285 nm and 255?270 nm, respectively.

Fig. 3. Oligomerization of dOtol1 (A) and hOtol1 (B) shown as the continuous sedimentation coefficient distribution at three protein concentrations in buffer F: 0.2 mg/ml, 0.5 mg/ml and 1.0 mg/ml. Panels C and D represent the dOtol1 and hOtol1 sedimentation coefficient distributions in the presence of 2 mM 2-mercaptoethanol (2MeEtOH).

Fig. 4. The dOtol1 (A and B) and hOtol1 (C and D) crosslinking assays were examined by SDS-PAGE and Western blotting.

Fig. 5. Negative stain transmission electron microscope images of dOtol1 (A) and hOtol1 (B) oligomers with magnified selected structures shown in squares below (side length of a square corresponds to 35 nm).

Fig. 6. Thermal denaturation curves of 4 ?M dOtol1 (A) and hOtol1 (B) in buffer F supplemented with 10 mM EDTA or a range of Ca2+ concentrations performed with nanoDSF.

Fig. 7. CD spectra of 10 ?M dOtol1 (A) and hOtol1 (B) recorded in buffer F supplemented with 10 mM EDTA or 1.0, 10 and 100 mM Ca2+ concentrations.

Fig. 8. Fluorescence emission spectra recorded for the ANS probe with 2 ?M dOtol1 (A) or hOtol1 (C) in the absence or presence of Ca²⁺ excited at 351 nm. The changes in the fluorescence increase and maximal intensity wavelength upon ANS binding to dOtol1 and hOtol1 are shown in B and D, respectively.

Fig. 9. The chromatograms of aSEC of 100 ?L (0.5 mg/ml) dOtol1 (A) and hOtol1 (B) in the presence of 10 mM EDTA or 10 mM Ca²⁺ in buffer E.

Fig. 10. The changes in $\overline{s_{20w}}$ derived from AUC of 0.2 mg/ml, 0.5 mg/ml and 1.0 mg/ml concentrations of dOtol1 (A) and hOtol1 (B) in the presence of 10 mM EDTA or 10 mM Ca²⁺. The sedimentation profile is shown in Fig. S4. Panel C represents the dependence of the cumulative radius of dOtol1 and hOtol1 on protein concentration and Ca²⁺ presence estimated with DLS. The fluorescence anisotropy values of ATTO-488-labelled dOtol1 and hOtol1 at increasing Ca²⁺ concentrations are shown in Panel D.