The structure of FSTL3.activin A complex. Differential binding of N-terminal domains influences follistatin-type antagonist specificity

Abstract

Transforming growth factor beta family ligands are neutralized by a number of structurally divergent antagonists. Follistatin-type antagonists, which include splice variants of follistatin (FS288 and FS315) and follistatin-like 3 (FSTL3), have high affinity for activin A but differ in their affinity for other ligands, particularly bone morphogenetic proteins. To understand the structural basis for ligand specificity within FS-type antagonists, we determined the x-ray structure of activin A in complex with FSTL3 to a resolution of 2.5 A. Similar to the previously resolved FS.activin A structures, the ligand is encircled by two antagonist molecules blocking all ligand receptor-binding sites. Recently, the significance of the FS N-terminal domain interaction at the ligand type I receptor site has been questioned; however, our data show that for FSTL3, the N-terminal domain forms a more intimate contact with activin A, implying that this interaction is stronger than that for FS. Furthermore, binding studies revealed that replacing the FSTL3 N-terminal domain with the corresponding FS domain considerably lowers activin A affinity. Therefore, both structural and biochemical evidence support a significant interaction of the N-terminal domain of FSTL3 with activin A. In addition, structural comparisons with bone morphogenetic proteins suggest that the interface where the N-terminal domain binds may be the key site for determining FS-type antagonist specificity.

FIGURE 1.

Domain architecture of FSTL3 and FS. FSTL3 lacks FSD3 and a heparin-binding sequence located within FSD1. FS315 and FSTL3 both have an extended acidic C-terminal tail. The percentage of identity is indicated for each domain excluding the conserved cysteines.

FIGURE 2.

Overall comparison of the FSTL3·activin A complex with FS288·activin A. Both FSTL3 and FS bind activin A in a similar fashion. a, two molecules of FSTL3 (transparent surface, orange and yellow) bind the central activin A homodimer as compared with the FS288·activin A structure in b. The third FS domain, which is not present in FSTL3, is colored gray. c, schematic depicting the blockade of both type I and type II receptor interfaces (N = ND, D1-3 = FSD1-FSD3). In FS, an interaction is observed between the N-terminal domain of one FS with FSD3 of the adjacent FS (noted by three cyan lines).

FIGURE 3.

Buried surface area differences on activin A at the type I interface. a, schematic depicting the distribution of buried surface area on each activin A subunit by interactions with the N-terminal domain of FSTL3 (yellow) and FS (red). The type I interface consists of both monomers (monomer 1, green; monomer 2, blue). FSTL3 buries more surface on monomer 1, whereas FS buries more surface on monomer 2 through interactions with the prehelix loop, which adopts a novel helix conformation termed α′. b, surface of activin A representing the difference in buried surface area for individual activin A atoms from interaction with FSTL3 and FS (ΔBSA = FSTL3_BSA - FS_BSA). The surfaces are colored with a three-step (yellow/white/red) gradient from 25 to -25 Ų. The yellow surfaces depict where FSTL3 buries more activin surface than FS, and the red surfaces depict where FS buries more than FSTL3. The white surfaces indicate that either no interaction occurs with antagonists, or the difference in buried surface area upon binding FSTL3 versus FS is minimal.

FIGURE 4.

Comparison of FSTL3 and FS N-terminal domain interactions with activin A. a and b, the N-terminal domain of FSTL3 is oriented more closely than that of FS to activin A monomer 1 (green). The prehelix loop region on activin A is disordered when bound to FSTL3, whereas in FS it forms the α′ helix (pink). There are also significant structural differences in the C-terminal loop segment on FSTL3/FS following the helix (cyan). c, superposition of only activin A monomer 1 in both complexes, which depicts the position of the each N-terminal domain relative to activin A. The N-terminal domain of FSTL3, including the helix, is closer to activin A by 3.5 and 2.9 Å, respectively. The distances were calculated from the center of mass (COM) of activin A backbone residues 25-28 to the COM of each N-terminal domain or the COM of each helix (FSTL3 residues 51-63 and FS residues 42-53). The asterisk indicates where FSTL3 would clash with the α′ of activin A from the FS structure. d, superposition of only the N-terminal domains. The FSTL3 domain is much more compact with reference to the main helix. The distance between the helices was measured from the COM of FSTL3 (residues 51-63) to the COM of FS (residues 42-53). All of the COM calculations were performed with the program CALCOM (50). In e and f, the structure is rotated 180° from a-c and shows differences in how the N-terminal helices interact with the two conserved tryptophan residues (at positions 25 and 28) of activin A. A significant difference in the orientation of Trp²⁸ is observed in the two structures; this permits Leu⁵⁷ of FSTL3 to wedge between Trp²⁵ and Trp²⁸ of activin A.

FIGURE 5.

Electron density of FSTL3 N-terminal domain interaction with activin A. Shown is a 2F_o - F_c electron density map contoured at 1.5 σ depicting the activin A tryptophan residues and their interaction with the N-terminal domain helix.

FIGURE 6.

Similarities between N-terminal domain and type I receptor ligand binding strategies. a, overall ligand binding position and domain size are similar among FSTL3/FS N-terminal domains and the BRIA type I receptor with a significant contribution to the binding interface coming from a single helix. b, side view of each domain with the common phenylalanine that is found at the ligand interface highlighted. The direction of the helix is indicated and runs in the reverse direction in BRIA. c, in each case a lysine, located in different regions of the helix, partially buries the conserved tryptophan residues.

FIGURE 7.

Structural differences between activin A and BMP may explain antagonist specificity. a and b, superposition of BMP2 and BMP7 onto the FSTL3·activin A complex, aligning only monomer 1 of activin A. The ribbon of activin A is shown, but for clarity, only the prehelix loop residues for each BMP (BMP2 (pink) and BMP7 (purple)) are depicted. The dotted lines indicate regions on BMP close enough to clash with the N-terminal domains. These appear more extensive for FSTL3. c, overall scheme for FSTL3 and FS binding to activin A and BMPs. We propose that the N-terminal domain of FS-type antagonists does not interact favorably with BMPs, thus accounting for the decreased affinity. We propose that the low affinity still observed for FS may be a result of the FS(ND)FS(FSD3) interactions and/or structural variation in the antagonist N-terminal domains.