Crystal structure of the WFIKKN2 follistatin domain reveals insight into how it inhibits growth differentiation factor 8 (GDF8) and GDF11

Abstract

Growth differentiation factor 8 (GDF8; also known as myostatin) and GDF11 are closely related members of the transforming growth factor ␤ (TGF-␤) family. GDF8 strongly and negatively regulates skeletal muscle growth, and GDF11 has been implicated in various age-related pathologies such as cardiac hypertrophy. GDF8 and GDF11 signaling activities are controlled by the extracellular protein antagonists follistatin; follistatin-like 3 (FSTL3); and WAP, follistatin/kazal, immunoglobulin, Kunitz, and netrin domain-containing (WFIKKN). All of these proteins contain a follistatin domain (FSD) important for ligand binding and antagonism. Here, we investigated the structure and function of the FSD from murine WFIKKN2 and compared it with the FSDs of follistatin and FSTL3. Using native gel shift and surface plasmon resonance analyses, we determined that the WFIKKN2 FSD can interact with both GDF8 and GDF11 and block their interactions with the type II receptor activin A receptor type 2B (ActRIIB). Further, we solved the crystal structure of the WFIKKN2 FSD to 1.39 Å resolution and identified surfaceexposed residues that, when substituted with alanine, reduce antagonism of GDF8 in full-length WFIKKN2. Comparison of the WFIKKN2 FSD with those of follistatin and FSTL3 revealed differences in both the FSD structure and position of residues within the domain that are important for ligand antagonism. Taken together, our results indicate that both WFIKKN and follistatin utilize their FSDs to block the type II receptor but do so via different binding interactions.

Characterization of tolloid-mediated cleavage of the GDF8 procomplex

“…Luciferase assays were conducted, in large part, as previously described (35,(53)(54)(55)(56)(57). In short, HEK293-T cells stably transfected with a firefly luciferase reporter gene under the control of the SMAD3-responsive (CAGA) 12 promoter were seeded in growth media at 20,000 cells per well in a 96-well, poly-D lysine coated plate.…”

Structural biology of the TGFβ family

“…Currently, the Kunitz 2 domain of WFIKKN1 and the FSD of WFIKKN2 have been solved by X-ray crystallography in the absence of ligand. 98,99 The Kunitz 2 domain resembles a typical Kunitz protease inhibitor, containing two antiparallel b-strands, an adjacent a-helix and a flexible loop that is predicted to insert into target proteases, namely trypsin. 98 The structure of WFIKKN2 FSD revealed a similar two domain architecture with an epidermal growth factor (EGF) and Kazal subdomain that is conserved in other FSDs.…”

“…However, the orientation of the WFIKKN2 FSD subdomains is unique from Fst FSDs 1-3, including FSD1 of Fst unbound to ligand. 99 Currently, the molecular mechanisms utilized by the multiple domains of WFIKKN to inhibit GDF8 and GDF11 is unknown, as no structure of the domains or full-length WFIKKN has been solved.…”

Exercise Partially Rejuvenates Muscle Stem Cells

“…Another possible serum actor conferring rejuvenative benefit is follistatin, which has been reported to increase after concurrent exercise (a combination of resistance and endurance training) in sarcopenic old men. 27 Follistatin binds to and antagonizes TGF-beta family members activin A, myostatin (GDF8), and GDF11, 28,29 leading to reduced inflammation, muscle hypertrophy, 30 when overexpressed, and importantly increased cyclin D1 expression at least in a subpopulation of MuSCs called myosphere-derived progenitor cells. 31 These observations raise the question whether follistatin is induced under the voluntary running conditions employed by Brett et al and whether follistatin then induces cyclin D in MuSC.…”