Crystal Structures of the Heparan Sulfate-binding Domain of Follistatin

Abstract

Follistatin associates with transforming growth factor-␤-like growth factors such as activin or bone morphogenetic proteins to form an inactive complex, thereby regulating processes as diverse as embryonic development and cell secretion. Although an interaction between heparan sulfate chains present at the cell surface and follistatin has been recorded, the impact of this binding reaction on the follistatin-mediated inhibition of transforming growth factor-␤-like signaling remains unclear. To gain a structural insight into this interaction, we have solved the crystal structure of the presumed heparan sulfate-binding domain of follistatin, both alone and in complex with the small heparin analogs sucrose octasulfate and D-myo-inositol hexasulfate. In addition, we have confirmed the binding of the sucrose octasulfate and D-myo-inositol hexasulfate molecules to this follistatin domain and determined the association constants and stoichiometries of both interactions in solution using isothermal titration calorimetry. Overall, our results shed light upon the structure of this follistatin domain and reveal a novel conformation for a hinge region connecting epidermal growth factor-like and Kazal-like subdomains compared with the follistatin-like domain found in the extracellular matrix protein BM-40. Moreover, the crystallographic analysis of the two protein-ligand complexes mentioned above leads us to propose a potential location for the heparan sulfate-binding site on the surface of follistatin and to suggest the involvement of residues Asn 80 and Arg 86 in such a follistatin-heparin interaction.The secreted polypeptide follistatin regulates several signaling pathways in a cell-and tissue-specific manner, largely through its ability to inactivate transforming growth factor-␤-like growth factor molecules (1, 2). Although first identified as a factor capable of countering the inducing effects of activin on follicle-stimulating hormone secretion (3, 4), follistatin has now been isolated and characterized from a variety of tissues and organisms, where it has been shown to take part in processes such as cell growth, differentiation, and secretion (5-7). Moreover, the importance of this polypeptide has been demonstrated in follistatin-null mice, which can survive birth but die from multiple skeletal and cutaneous abnormalities within a few hours of delivery (8).Follistatin isoforms sharing the same N terminus but differing in the length of their polypeptide chain and in their glycosylation patterns coexist in vivo, with molecular masses ranging from 31 to 39 kDa (4, 9). Indeed, alternative splicing of the follistatin gene yields the Fs-315 and Fs-288 isoforms, featuring 315 and 288 residues, respectively. An additional variant of follistatin, Fs-303, is thought to arise from the proteolytic cleavage of the Fs-315 C terminus and is likely to be the predominant species in vivo (3, 10, 11). Despite its relatively limited size, follistatin contains a total of 36 cysteine residues, believed to be arranged into nonoverlapping set...

Potential inhibitors of chemokine function: Analysis of noncovalent complexes of cc chemokine and small polyanionic molecules by ESI FT-ICR mass spectrometry

“…Sucrose octasulfate (SOS, Scheme 1b) has been shown to bind to many heparin-binding proteins including follistatin [36], fibroblast growth factor (FGF) [37], and hepatocyte growth factor [38]. Noncovalent complex ions were observed (Figure 1c) after a mixture containing 40 M MCP-1 and 200 M SOS was subjected to the filtration trapping assay.…”

Structural and Biophysical Coupling of Heparin and Activin Binding to Follistatin Isoform Functions

“…Mutations in this region have identified lysines 75, 76, 81, and 82 as critical for cell surface association (38). Asparagine 80 and arginine 86 have also been shown to be important in binding to small heparin analogs (39). The affinity differences in cell surface association between FS315, -303, and -288 have been attributed to the highly acidic nature of the C-terminal amino acids present in the longer isoforms of follistatin, where 11 of 13 contiguous residues are either glutamic acid or aspartic acid in FS315.…”

Solution Structure of Factor I-like Modules from Complement C7 Reveals a Pair of Follistatin Domains in Compact Pseudosymmetric Arrangement

“…The FOLN subdomain within the FDs commonly contains a long loop of seven or more amino acid residues connecting the two ␤-strands that comprise the ␤-hairpin, whereas this loop is reduced to two and four residues in FIM1 and FIM2, respectively. In follistatin, this loop provides a binding site for heparin (65), and it is also implicated in ligand binding in other FDs (71). In addition, the C7-FIMs have an insertion between B3 and B4 within the KAZAL subdomain, effectively lengthening strand B3; in FIM1, this insertion also extends the interstrand loop by two basic amino acid residues.…”

“…It is thus noteworthy that FIM1 exposes a predominantly electropositive face, including the conserved lysine at position 707, within the context of C7-FIMs FIM1 and FIM2), green (FIM1 only), and yellow (FIM2 only) squares (determined using GETAREA (50)). c, domain cartoon representations of C7, follistatin fragments: 1LR7, 1LR8, 1LR9 (65), and 2ARP (73); full-length follistatin: 2P6A (75) and 1B0U (76); follistatin like protein: 3B4V (74); and SPARC: 1NUB (64), 1BMO (71), and 2V53 (72). Where more than one FD was present in the Protein Data Bank, each is consecutively numbered.…”