Analysis of human follistatin structure: Identification of two discontinuous N-terminal sequences coding for activin A binding and structural consequences of activin binding to native proteins

A primary physiological function of follistatin is the binding and neutrali zation of activin, a transforming growth factor-beta family growth factor, and loss of function mutations are lethal. Despite the critical biological importance of follistatin's neutralization of activin, the structural basis of activin's binding to follistatin is poorly understood. The purposes of these studies were 1) to identify the primary sequence(s) within the N-term inal domain of the follistatin coding for activin binding, and 2) to determ ine whether activin binding to the native protein causes changes in other s tructural domains of follistatin. Synthetic peptide mimotopes identified within a 63-residue N-terminal domai n two discontinuous sequences capable of binding labeled activin A. The fir st is located in a region (amino acids 3-26) of follistatin, a site previou sly identified by directed mutagenesis as important for activin binding. Th e second epitope, predicted to be located between amino acids 46 and 59, is newly identified. Although the sequences 3-26 and 46-59 code for activin b inding, native follistatin only binds activin if disulfide bonding is intac t. Furthermore, pyridylethylation of Cys residues followed by N-terminal se quencing and amino acid analysis revealed that all of the Cys residues in f ollistatin are involved in disulfide bonds and lack reactive free sulfhydry l groups. Specific ligands were used to probe the structural effects of activin bindi ng on the other domains of the full-length molecule, comprised largely of t he three 10-Cys follistatin module domains. No effects on ligand binding to follistatin-libe module I or II were observed after the binding of activin A to native protein. In contrast, activin binding diminished recognition o f domain III and enhanced that of the C domain by their respective monoclon al antibody probes, indicating an alteration of the antigenic structures of these regions. Thus, subsequent to activin binding, interactions are Likel y to occur between regions of follistatin located in different domains and separated by considerable lengths of linear sequence. Such interactions cou ld have important functional significance with respect to the structural he terogeneity of naturally occurring follistatins.