Characterization of the structural features and interactions of sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation

Abstract

The secreted glycoprotein sclerostin has recently emerged as a key negative regulator of Wnt signaling in bone and has stimulated considerable interest as a potential target for therapeutics designed to treat conditions associated with low bone mass, such as osteoporosis. We have determined the structure of sclerostin, which resulted in the identification of a previously unknown binding site for heparin, suggestive of a functional role in localizing sclerostin to the surface of target cells. We have also mapped the interaction site for an antibody that blocks the inhibition of Wnt signaling by sclerostin. This shows minimal overlap with the heparin binding site and highlights a key role for this region of sclerostin in protein interactions associated with the inhibition of Wnt signaling. The conserved N- and C-terminal arms of sclerostin were found to be unstructured, highly flexible, and unaffected by heparin binding, which suggests a role in stabilizing interactions with target proteins.

FIGURE 1.

Inhibition of Wnt3a-dependent signaling by recombinant sclerostin produced in mammalian cells and E. coli. MC3T3-E1 cells stably transfected with a Super-TOPFlash reporter construct were treated with Wnt3a (50 ng/ml) and purified human recombinant sclerostin expressed either in mammalian cells or E. coli. The data shown illustrate the comparable inhibition of Wnt-dependent signaling obtained with either mammalian cell-derived sclerostin (black bars) or E. coli-derived ¹⁵N-labeled sclerostin (white bars).

FIGURE 2.

Solution structure of sclerostin. Panel A shows a best-fit superposition of the protein backbone for the family of 36 converged structures obtained for sclerostin, whereas panel B contains a ribbon representation of the backbone topology of the structured core of the protein (residues 52 to 147) in the same orientation. Panel C shows a schematic representation of the protein, highlighting the positions of disulfide bonds, the resulting 3 loop structure, and residues involved in regular β-sheets (blue).

FIGURE 3.

Sequence conservation between structural homologues in the cystine-knot family. The figure shows an optimized structure-based sequence alignment for the structured region of sclerostin against structurally related members of cystine-knot family. The highly conserved cysteines involved in formation of the cystine-knot motif are shaded in pink, and conserved or semi-conserved hydrophobic residues are highlighted in blue. The residue numbers and positions of regular secondary structure elements shown above the sequence alignment refer to sclerostin. The multiple alignment was produced with JalView (33) using an optimized structure based alignment determined by DALI (46). PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor; TGF, transforming growth factor; GDNF, glial cell line-derived neurotrophic factor; COAG, coagulation factror; BDNF, brain-derived neurotrophic factor; CG, chorionic gonadotropin.

FIGURE 4.

Surface features of sclerostin. Panels A and B show contact surface views of sclerostin, which are colored according to electrostatic potential, with areas of significant positive charge shown in blue, significant negative charge in red, and neutral in white. The orientation of the protein in panel A is equivalent to the ribbon representation in panel C. The location of the hydrophobic patch on the concave surface of the extended finger-like structures is indicated by the arrow. Panels C and D show a ribbon representation of sclerostin, with the positions of the basic side chains from arginine and lysine residues highlighted.

FIGURE 5.

Characterization of the interaction of heparin with sclerostin. Panel A shows an overlay of selected regions from ¹H,¹⁵N HSQC spectra of uniformly ¹⁵N-labeled sclerostin (200 μm) acquired in the absence (blue) and presence (red) of an equimolar concentration of heparin 12-mer. Changes in the positions of individual backbone amide peaks illustrate the shifts induced by heparin binding, which are summarized by the histogram of backbone amide chemical shift change versus sequence shown in panel B. Panel C shows a surface view of sclerostin, in which residues are colored according to the perturbation of their backbone amide signals induced by heparin binding, as indicated by the scale below. The protein is shown in a similar orientation to the ribbon representation contained in Fig. 2. Panel D shows a representative view of heparin docked onto its binding site on the surface of sclerostin using ambiguous interaction restraints derived from the NMR data.

FIGURE 6.

Localization of sclerostin to the surface of cells. The top section of panel A shows a Western blot (with an antibody to sclerostin) of 24-h supernatants obtained from MC3T3-E1 cells transfected with wild type human sclerostin. The transfections and treatments were as follows: 1, empty vector; 2, sclerostin; 3, empty vector with heparin added to a final concentration of 500 μg/ml; 4-12, sclerostin with heparin added to final concentrations of 500, 250, 100, 50, 25, 12.5, 1, 0.5, and 0.25 μg/ml, respectively. The bottom section of panel A shows the Western blot of samples generated from corresponding wells incubated with a lysis buffer to determine the total amount of sclerostin being produced. The top section of panel B shows the Western blot (with an antibody to sclerostin) obtained for samples of 24-h supernatants from MC3T3-E1 cells transfected with vectors encoding wild type or mutant sclerostin. The transfections were as follows: 1, empty vector; 2, wild type sclerostin; 3, sclerostin R114A, R116A, and R119A; 4, sclerostin K134A and R136A; 5, sclerostin R97A, K99A, and R102A; 6, sclerostin K142A, K144A, and R145A. The bottom section of panel B shows the Western blot obtained for equivalent samples of total cell lysates.

FIGURE 7.

Blocking of the inhibition of the Wnt/β-catenin signaling pathway by sclerostin. Panel A shows the luciferase activity of MC3T3-E1 cells stably transfected with a Super-TOPFlash reporter construct, which were treated with Wnt3a conditioned medium and ¹⁵N-labeled human recombinant sclerostin (2 μg/ml) in the presence or absence of Scl-AbI or an isotype-matched control antibody (Ab, both 100 μg/ml). Data are presented as the mean ± S.E. (n = 8). Statistical analysis was carried out by one way analysis of variance with a Bonferroni post hoc test (^***, p < 0.001). Panel B shows the effects of Scl-AbI on total area bone mineral density (BMD) of mice as measured by DEXA scans. The antibody was administered (25 mg/kg subcutaneously (s.c.)) at either days 0 or 42 (triangles) or once every 2 weeks throughout the experiment (squares). Results obtained from animals treated with a phosphate-buffered saline (PBS) control alone are shown for comparison (circles). Data are presented as the mean ± S.E. (n = 8-10). Statistical analysis was carried out by one way analysis of variance with a Bonferroni post hoc test (^***, p < 0.001). d, day.

FIGURE 8.

Identification of the binding site of an antibody that blocks the sclerostin-mediated inhibition of Wnt signaling. Panel A shows selected regions from overlaid ¹H,¹⁵N HSQC spectra of uniformly ¹⁵N labeled sclerostin (200 μm) acquired in the absence (blue) and presence (red) of an equimolar amount of an antibody (Fab fragment of Scl-AbI) known to block the inhibition of Wnt signaling by sclerostin. The minimal backbone amide chemical shift changes observed for sclerostin on Fab binding are summarized in the histogram shown in panel B. Panel C contains a surface view of sclerostin in which residues are colored according to the perturbation of their backbone amide signals induced by Fab binding, as indicated by the scale below. The orientation of the protein in panel C is equivalent to the ribbon representation in Fig. 2.