Fig. 9

SCUBE3-N294K mutant has a defective interaction with the BMP type IA receptor, diminishing osteogenic BMP signaling. A–B Close-up of SCUBE3 cbEGF₇ domains in N294 and N294K mutant. 3-D structure of mouse SCUBE3 seventh EGF-like domain (D277 to C316) was extracted from the AlphaFold2 model (AF-Q66PY1-F1-model_v3) [187]. Calcium ion (green sphere) was taken from the fifth EGF-like domain of human NOTCH1 (PDB 5FM9) [221] and coordinated by the oxygen atoms of D277, E280 and N294 side-chains and carboxyl atoms of I278, T295 and S298 backbones (shown as pink dotted lines with carbon atoms colored in yellow, oxygen atoms in red, and nitrogen atoms in blue). Additionally, hydrogen bonds (red dotted lines) form between the nitrogen atom of the N294 side-chain and the carboxyl atom of the D277 side chain and between the hydroxyl atom of D277 and the backbone nitrogen of G297 to stabilize the overall conformation of the cbEGF₇ domain (A). For the N294K mutant, the lysine residue is shown in ‘stick-mode’ with carbon atoms colored in cyan. The mutant K294 forms a hydrogen-bond network (red dotted lines) with oxygen atoms from the side-chains of D277 and E280 and backbone of I278. Of note, the nitrogen of K294 possibly excludes and occupies the position of calcium. This figure was produced using PyMOL (Schrödinger, LLC. The PyMOL molecular graphics system, version 1.8, https://pymol.org) (B). C Protein domain structure depicting the amino acid exchange of N294K lying within the calcium-binding seventh EGF-like domain (cbEGF₇) of SCUBE3. D Cell-surface distribution of SCUBE3-wild-type (WT) and -N294K protein. The expression plasmids encoding FLAG-tagged SCUBE3-WT or -N294K were transfected in HepG2 cells for 2 days. Immunofluorescence staining of cells with anti-FLAG (red) antibody. Nuclei were visualized by DAPI staining (blue). Scale bar = 10 μm. E SCUBE3-N294K mutation blunts BMP2/4-stimulated signaling activity. The BMP-responsive luciferase reporter (BRE-luc) and pRL-TK alone or in combination with the designated expression vectors were transfected into HepG2 cells. Luciferase activity was assessed 24 h after transgenic cells were treated with or without BMP2 or BMP4 (50 ng/ml). Firefly luciferase values were normalized to Renilla activity to show relative luciferase activity. The experiments were performed 3 times in triplicate. Data are mean ± SD. **p < 0.01. F Effect of the SCUBE3-N294K mutant on interactions with BMP signaling cascade receptors. In HEK-293T cells, HA-tagged BMPR-IA, HA-tagged BMPR-IB, or Myc-tagged BMPR-II constructs were co-transfected with plasmids expressing FLAG-tagged SCUBE3-WT or -N294K. In order to identify protein–protein interactions, transfected cell extracts were immunoprecipitated after 48 h and used for western blot analysis with the designated antibodies