Structural characterization of an activin class ternary receptor complex reveals a third paradigm for receptor specificity Erich J. Goebela, Richard A. Corpinab, Cynthia S. Hinckc, Magdalena Czepnika, Roselyne Castonguayd, Rosa Grenhad, Angela Boisvertd, Gabriella Miklossye, Paul T. Fullertone, Martin M. Matzuke, Vincent J. Idoneb, Aris N. Economidesb, Ravindra Kumard, Andrew P. Hinckc, and Thomas B. Thompsona,1 aDepartment of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, Cincinnati, OH 45267; bSkeletal Diseases Therapeutic Focus Area, Regeneron Pharmaceuticals, Tarrytown, NY 10591; cDepartment of Structural Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260; dDiscovery Group, Acceleron Pharma, Cambridge, MA 02139; and eDepartment of Pathology and Immunology, Baylor College of Medicine, Houston, TX 77030 Edited by K. Christopher Garcia, Stanford University, Stanford, CA, and approved June 24, 2019 (received for review April 18, 2019) TGFβ family ligands, which include the TGFβs, BMPs, and activins, I receptors with higher affinity than TGFβ class ligands (5, 6). signal by forming a ternary complex with type I and type II recep- Structural studies describing ligand–receptor interactions have tors. For TGFβs and BMPs, structures of ternary complexes have revealed how TGFβ and BMP ligands adopt different strategies to revealed differences in receptor assembly. However, structural in- assemble receptors, accounting for the differences in type I affinity formation for how activins assemble a ternary receptor complex is (7–11). The ternary structure of BMP2 revealed independent re- lacking. We report the structure of an activin class member, ceptor binding sites, with the type II receptors binding on the GDF11, in complex with the type II receptor ActRIIB and the type convex “knuckle” region of the ligand and the type I receptors I receptor Alk5. The structure reveals that receptor positioning is binding through extensive contacts in the concave cleft formed by similar to the BMP class, with no interreceptor contacts; however, the “wrist helix” at the dimer interface (12). In stark contrast, the type I receptor interactions are shifted toward the ligand fin- receptor binding for TGFβ ligands uses a cooperative assembly gertips and away from the dimer interface. Mutational analysis mechanism that facilitates binding of the type I receptor. Here, the BIOCHEMISTRY shows that ligand type I specificity is derived from differences in type II receptor is shifted toward the ligand fingertips and comes the fingertips of the ligands that interact with an extended loop into direct contact with the type I receptor; thus, binding of the specific to Alk4 and Alk5. The study also reveals differences for type I receptor is significantly stronger when the ligand is pre- how TGFβ and GDF11 bind to the same type I receptor, Alk5. For occupied with the type II receptor (9, 10). These studies highlight GDF11, additional contacts at the fingertip region substitute that different mechanisms of ligand–receptor interactions have for the interreceptor interactions that are seen for TGFβ, indicating emerged within the TGFβ family. that Alk5 binding to GDF11 is more dependent on direct contacts. In support, we show that a single residue of Alk5 (Phe84), when mutated, abolishes GDF11 signaling, but has little impact on TGFβ Significance signaling. The structure of GDF11/ActRIIB/Alk5 shows that, across the TGFβ family, different mechanisms regulate type I receptor bind- The TGFβ family of ligands plays fundamental roles in con- ing and specificity, providing a molecular explanation for how the trolling diverse signaling events throughout multicellular or- activin class accommodates low-affinity type I interactions without ganisms. The family is divided into 3 classes: activin, BMP, and the requirement of cooperative receptor interactions. TGFβ, with each signaling through specific combinations of type I and type II receptors. We demonstrate that activin li- GDF11 | TGF-β superfamily | activin | ternary signaling complex | Alk5 gands bind receptors similarly to BMP ligands, but they utilize the ligand fingertip to make specificity-determining contacts he transforming growth factor β (TGFβ) family of ligands is with the type I receptors. We also show that different molec- β Tcomposed of more than 30 dimeric growth factors essential in ular mechanisms have emerged for how GDF11 and TGF bind the development and homeostasis of all animals (1–3). Signaling the same type I receptor, whereby GDF11 is more dependent occurs when ligands engage 2 type I and 2 type II serine/threo- on direct receptor contacts. Thus, this study provides insight at nine kinase receptors, forming a hexameric signaling complex (4, the molecular level into how the activin class ligands bind and 5). Both the type I and type II receptors consist of a small ex- assemble their type I and type II receptors. tracellular ligand-binding domain tethered to an intracellular Author contributions: E.J.G., R.A.C., V.J.I., A.N.E., and T.B.T. designed research; E.J.G., kinase domain. Upon assembly, type II receptors phosphorylate R.A.C., M.C., V.J.I., and A.N.E. performed research; C.S.H., R.C., R.G., A.B., G.M., P.T.F., and activate the type I receptors, which, in turn, phosphorylate M.M.M., R.K., and A.P.H. contributed new reagents/analytic tools; E.J.G., R.A.C., V.J.I., intracellular SMAD proteins. Signaling is then transduced into the A.N.E., and T.B.T. analyzed data; and E.J.G. and T.B.T. wrote the paper. nucleus, where activated SMADs act as transcriptional factors, Conflict of interest statement: T.B.T. is a consultant for Acceleron Pharma and Scientific enabling signaling outcomes dependent on ligand–receptor com- Founder for Eclode. R.C. and R.K. are current employees of Acceleron Pharma with ownership interest in the company. A.N.E. and V.J.I. are current employees of Regeneron Pharmaceuticals binations. Given the diversity of biological responses and pro- with ownership interest in the company. The other authors report no competing cesses controlled by the family, it is somewhat surprising that only interests. 7 type I and 5 type II receptors are found within mammals (1). This article is a PNAS Direct Submission. Based on sequence homology and receptor utilization, the Published under the PNAS license. β TGF family can be divided into 3 general classes of ligands: Data deposition: The ternary structure of GDF11 bound to ActRIIB-ECD and Alk5-ECD activins, BMPs, and TGFβs. From this, 2 general signaling par- described in this paper has been deposited to the Protein Data Bank, http://www.rcsb. adigms have emerged—the TGFβ class, which activates SMAD org/ (accession code 6MAC). 2/3; and the BMP class, which activates SMAD 1/5/8—with 1To whom correspondence may be addressed. Email: [email protected]. specificity for each paradigm dependent on the activation of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. different type I receptors. Interestingly, ligands have different 1073/pnas.1906253116/-/DCSupplemental. affinities for type I receptors, with BMP class ligands binding type Published online July 17, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1906253116 PNAS | July 30, 2019 | vol. 116 | no. 31 | 15505–15513 Downloaded by guest on September 30, 2021 The activin class, which includes activin A (ActA), activin B (ActB), Growth and Differentiation Factor 8 (GDF8), and GDF11, shares aspects of both the TGFβ and BMP signaling paradigms. Similar to TGFβ, activin class ligands bind type II receptors with high affinity, including ActRIIA and ActRIIB, and type I receptors with very low affinity, to activating SMAD2/ 3 (1). However, type II receptor positioning for the activin class is similar to BMP ligands, suggesting that a cooperative mecha- nism between type I and type II receptors is not used by activin ligands to facilitate binding of the low-affinity type I receptors, as is the case for TGFβ (13, 14). Whereas TGFβ primarily signals use the type I receptor Activin receptor-like kinase 5 (Alk5), the activin class ligands are more promiscuous and can use different combinations of the type I receptors: Alk4, Alk5, and Alk7 (15– 17). For example, whereas ActA predominantly signals using Alk4, ActB can signal effectively using both Alk4 and Alk7 (5, 18, 19). GDF8 and GDF11 can both signal using Alk4 and Alk5, with GDF11 extending specificity to Alk7 (17). Although struc- tural studies have revealed how the TGFβ and the BMP ligands assemble the type I and type II receptors, the molecular details for how activin class ligands form a ternary signaling complex and account for type I receptor specificity is unknown. To ad- dress this, we sought to structurally characterize a ternary com- plex of an activin class ligand. Results and Discussion Ternary Complex Structure of GDF11/ActRIIB-ECD/Alk5-ECD. Crystals of an activin class ternary complex were generated by combining Fig. 1. Structure of GDF11/ActRIIB/Alk5 ternary complex. (A) GDF11/ActRIIB/ GDF11 and the ectodomains (ECDs) of both the high-affinity Alk5 as viewed on the membrane surface. GDF11 has 2 monomers repre- type II receptor ActRIIB and the low-affinity type I receptor sented in slate (monomer A) and cyan (monomer B). ActRIIB-ECD is repre- Alk5. Diffraction data were collected to 2.3-Å resolution, and the sented in orange, with Alk5-ECD in yellow. (B) Ninety-degree upward view structure was solved by molecular replacement using the previ- of the ternary complex. ActRIIB binds at the convex knuckle region of ously determined individual components as search models (data GDF11 whereas Alk5 binds at the concave interface of GDF11 formed be- collection and refinement statistics are provided in SI Appendix, tween the fingertip of monomer A and the wrist helix of monomer B GDF11.