The crystal structure of the activin B:Fst288 complex and computational insights into the broad antagonistic activity and specificity of follistatin.

Members of the transforming growth factor-β (TGF-β) family regulate essential biological processes, and their activity is tightly controlled by extracellular antagonists like follistatin 288 (Fst288). While Fst288 potently inhibits several ligands, including activins A and B, GDF8, and GDF11, the structural basis for its interaction with activin B (ActB) has remained largely uncharacterized. This lack of data has limited our understanding of how Fst288 achieves such broad inhibitory activity while maintaining ligand-specific selectivity. In this study, we resolved the crystal structure of the ActB:Fst288 complex at 2.7 Å resolution. Our findings reveal that while Fst288 utilizes a conserved receptor-blocking mechanism, ActB engages the antagonist through a modified structural mode. Most notably, the fingertips of ActB form direct, unique contacts with the third follistatin domain (FSD3). This interaction disrupts the intermolecular head-to-tail cooperativity typically seen in Fst288 dimers, shifting the stabilization of the complex toward individual ligand-domain affinities. Computational analysis supports a model where ActB relies more on direct contacts with Fst288, whereas ActA relies on the intermolecular head-to-tail Fst288 interactions. Further computational analysis indicates ActB is more flexible than ActA and GDF8. These results suggest that Fst288's broad potency arises from a dynamic interplay between ligand flexibility and antagonist conformational plasticity that can accommodate different ligand surfaces. By elucidating unique characteristics of the ActB:Fst288 interface, this study deepens the understanding of ligand selectivity and provides a framework for the rational design of targeted TGF-β antagonists.