Structural and functional study of FK domain of Fstl1

Abstract

Fstl1 is a TGF-β superfamily binding protein which involved in many pathological processes. The function of Fstl1 has been widely elucidated, but its structural characterization has not been explored. Here we solved the high-resolution crystal structure of FK domain of murine Fstl1, analyzed its unique characteristics, and investigated its contribution to the function of full-length Fstl1. We found that Fstl1-FK forms a stable dimer in both solution and crystal, which suggest that this protein may function as a dimer during its interaction with TGF-β, a molecule known to form dimer during activation process. We also found this FK domain is indispensable for the proper function of Fstl1 during the transduction of TGF-β signaling. These observations provide important insights into the understanding of Fstl1 and may facilitate the exploration of this molecule in clinical study.

Keywords: FK domain; Fstl1; TGF-β signaling; crystal structure; lung fibrosis.

Figure 1

The overall structure of Fstl1‐FK domain. (a) Diagrammatic representation of Fstl1. Fstl1 contains FOLN and Kazal domain at N‐terminus, with two‐EF‐hand domain in the middle and VWFC domain at C‐terminus. (b) Overall structure of Fstl1‐FK domain. Fstl1‐FK domain consists of FOLN domain (green) and Kazal domain (orange). (c) Disulfide bonds. Disulfide bonds stabilize the overall structure of Fstl1‐FK domain. Five pairs of highly conserved disulfide bonds are highlighted by yellow sticks, and cysteines are labeled in dark blue color. (d) Salt bridges on the molecular surface. Glutamic acid and aspartic acid are colored in purple, arginine and lysine are colored in red. The numbers in the labels correspond to the amino acid sequence

Figure 2

Dimerization of Fstl1‐FK domain. (a) Ribbon representation of the dimer formed in the crystals. (b) The dimer is stabilized by two interactions: One from β‐sheets in FOLN domain (orange rectangle), the other from N‐terminal peptide and Kazal domain (red rectangle). (c) The results of size exclusion chromatography of Follistatin‐FS domain and Fstl1‐FK domain. Samples were loaded on Hiload 16/60 Superdex 200 column for purification. Orange line represents Follistatin‐FS domain, blue line represents Fstl1‐FK domain. Eluted fractions were also detected by SDS‐PAGE. M represents molecular mass standards. P1 and P2 means Peak 1 and Peak 2 on size exclusion column; Inj (injection) means the sample before loaded onto column. (d) Analytical ultracentrifuge analysis of Fstl1‐FK domain and Follistatin‐FS domain. Sedimentation coefficient distribution of the fractions from size exclusion column was shown. Peak 1 (green) and Peak 2 (blue) of Fstl1‐FK domain were detected as dimer and monomer respectively. For Follistatin‐FS domain, only Peak 2 was measured (red), since Peak 1 precipitates easily during AUC experiment. (e) Co‐immunoprecipitate of GFP‐Fstl1‐FK and HA‐Fstl1‐FK. Two proteins were co‐expressed in 293T cells and pulled‐down by GFP‐trap beads, then detected by anti‐HA antibody

Figure 3

Structural comparison between Fstl1‐FK domain and other proteins. (a) The amino acid sequence alignment of Fstl1‐FK with the other four proteins which have similar structures. The secondary structure elements are shown above the alignment. Conserved residues are highlighted in red. The green number below the alignment represents the conserved cysteines. (b) Superimposition of Fstl1‐FK domain (red) with BM‐40 FS domain (blue, PDB code 1BMO). (c) Superimposition of Fstl1‐FK domain (red) with Follistatin FS domain (blue, PDB code 1LR9). (d) Superimposition of Fstl1‐FK domain (red) with human complement factor I (blue, PDB code 2XRC). (e) Superimposition of Fstl1‐FK domain (red) with Fstl3 FS domain (blue, PDB code 2KCX)

Figure 4

The possible binding specificity of FK domains. (a) Superimposition of Fstl1‐FK domain (red) with free BM‐40 (green, PDB code 1BMO) and BM‐40‐collagen complex (PDB code 2V53, blue for BM‐40 and magenta for collagen). The loop region linking N‐terminal β‐sheet and kazal region was colored by yellow. Red arrow indicated the region with dramatic conformational shift. (b) The differences of electrostatic potential distribution between Fstl1‐FK domain (red) and BM40 (green). The different regions were labeled in the blue rectangles. (c) Superimposition of Fstl1‐FK domain (red) with Follistatin‐heparin analogs (green, PDB code 1LR7). (d) The differences of electrostatic potential distribution between Fstl1‐FK domain (red) and Follistatin (green, PDB code 1LR9). The different regions were labeled in the blue rectangles

Figure 5

FK domain is essential for Fstl1 in promoting TGF‐β1 signaling in lung epithelial cell and mouse embryonic fibroblasts (MEF). (a) Schematic representation of myc and His‐tagged full‐length Fstl1 (1–306 aa), FK domain deletion mutant and only FK domain mutant. (b) A549 cells were transfected with empty vector or Fstl1, Fstl1‐△FK and Fstl1‐FK plasmid for 48 hr, then treated with TGF‐β1 (5 ng/mL) for 48 hr. Cell lysates were immunoblotted with N‐cadherin and β‐actin (loading control throughout) antibodies. Shown were representative blots from three independent experiments. (c, d), A549 cells (c) or MEF cells (d) were treated with TGF‐β1 (5 ng/mL) and Fstl1 (100 ng/mL) or Fstl1‐△FK mutant protein (100 ng/mL) for 48 hr or 24 hr. Immunoblotting was performed to detect the protein levels of N‐cadhein, α‐SMA and β‐Actin. Shown were representative blots from three independent experiments

Figure 6

Fstl1 deletion mutant protein attenuates lung fibrosis after bleomycin challenge in vivo. (a) C57BL/6J mice were subjected to intratracheal injection of 2.5 U/kg bleomycin treatment, then intravenous administration of PBS or Fstl1‐△FK protein (2 μg/mouse) at 5, 8, 11 day. (b) H&E (a, b) and masson trichrome staining of collagen (c, d) on lung sections from PBS or Fstl1‐△FK protein injection mice after bleomycin treatment. Representative images of the staining are shown (n = 4 per group). (c) Hydroxyproline contents in lung tissues from PBS or Fstl1‐△FK protein injection mice after bleomycin treatment were measured (n = 4 per group; *, p < .05). (d) Mouse lung tissues were harvested for western blot analysis. Immunoblotting was performed to detect the protein levels of α‐SMA and β‐actin (n = 4 per group), three repetitive experiments were shown for each group