Abstract
Follistatin associates with transforming growth factor-beta-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-beta-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 Asn80 and Arg86 in such a follistatin-heparin interaction.
Highlights
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
Refined temperature factors for all three structures are highest for this N-terminal domain and for the extended coil in particular, a phenomenon observed in the BM-40 structure and most probably caused by the poor packing of loop 64 –71 against the small -hairpin formed by strands 1 and 2
In this paper we have presented the structure of Fs1, the putative heparan sulfate-binding domain of follistatin, both alone and in complex with the small heparin analogs sucrose octasulfate (SOS) and Ins6S
Summary
All of the chemicals used were of research grade or higher and were purchased from BDH, Difco, Fluka, Melford Laboratories, or Sigma, unless specified otherwise. Ins6S and SOS, both as potassium salts, were purchased from Sigma and Toronto Research Chemicals, respectively. Phases were extended to 1.5-Å with ARP/wARP [28] using data collected from a single native Fs1/SOS crystal at the ESRF beamline ID-14-4. Once a suitable model of Fs1 was obtained, its atomic coordinates were used to compute initial phases for the Fs1/Ins6S and Fs1 structures. Molecular replacement and rigid body refinement were performed in CNS, using the native data sets collected for Fs1/Ins6S and TABLE I Crystallographic statistics for the Fs1, Fs1/SOS, and Fs1/Ins6S structures. SOS and Ins6S in powder form were weighed out accurately and solubilized in the dialysis buffer to the final concentrations of 0.2 and 0.4 mM, respectively. Possible interaction of potassium (the counter ion for both Ins6S and SOS) with FS1 domain was studied but found to have no additional effect compared with the buffer alone control
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