Structural characterization of an activin class ternary 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 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 . 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 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 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

www.pnas.org/cgi/doi/10.1073/pnas.1906253116 PNAS Latest Articles | 1of9 Downloaded by guest on September 26, 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 -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. Table S1). Within the lattice, the asymmetric unit contains 1 GDF11 monomer bound to 1 ActRIIB-ECD and 1 Alk5-ECD with the gesting the absence of conformational changes upon receptor full complex being related through crystallographic symmetry. β Crystal contacts are exclusively through receptor contacts from binding (20). As observed for the BMP and TGF ternary com- symmetry-related complexes (SI Appendix,Fig.S1). The ternary plexes, the C-terminal ends of the receptors are oriented in the structure shows ActRIIB binding at the knuckle regions of GDF11 same direction, consistent for a cell surface signaling complex B and Alk5 binding in the concave clefts formed at the interface of (Fig. 1 ). the GDF11 dimer (Fig. 1). As expected, both ActRIIB and Alk5 adopt the conserved 3-finger toxin fold with slight variation Molecular Determinants of Receptor Specificity for the Activin Class. in antiparallel β-strandlength(SI Appendix, Fig. S2). Electron Next, we wanted to determine if the ternary complex of density was well defined for the following residues: GDF11 (300– GDF11 could provide insight into receptor specificity across the 407), ActRIIB (26–120), and Alk5 (33–112). Two previously dis- broader activin class. We first compared interactions of GDF11 ordered loops within Alk5 were resolved: the loop from β3toβ4 at the type II interface with previously determined structures, and the loop connecting α-helix1 to β5 (9). All residues at the in- including ActA/ActRIIB (13). Although the position of the type terfaces between each component were well defined in electron II receptor is consistent with previous structures, GDF11 forms more extensive contacts throughout the interface. This includes a density. 334 94 Overall, receptor assembly resembles that of the BMP para- unique salt bridge between Lys in GDF11 and Glu in ActRIIB SI Appendix digm, with each receptor binding independently and in compa- that is not present in the ActA/ActRIIB complex ( ,Fig. rable locations (7, 8). Two ActRIIB molecules bind on the S3 B and C) (8, 13). In total, GDF11 buries 12.5% more surface knuckle segment of the GDF11 fingers, similar to that observed area at the type II interface than ActA. Analysis of the interface for ActA and BMP9 (11, 13). Here, ActRIIB forms a concave stability in jsPISA reveals that the GDF11/ActRIIB interface is surface with a hydrophobic core, anchored by a triad of aromatic more energetically favorable with a ΔG P value of 0.2, compared residues (Tyr60,Trp78,andPhe101), that buries a total of 720 Å2 per with a value of 0.4 for both ActA and BMP2 (21). Further com- receptor (Fig. 2A and SI Appendix,Fig.S3A and B). Each parison of ActRIIA versus ActRIIB revealed potential differences ActRIIB interacts exclusively with 1 GDF11 monomer. In con- in how GDF11 binds to each type II receptor. For example, trast, Alk5 contacts both GDF11 monomers by binding in the ActRIIA would not form the aforementioned salt bridge because concave interface formed at the dimer interface. Here, the β4– Glu94 of ActRIIB is a lysine in ActRIIA (SI Appendix,Fig.S3B β5 loop and the α1 helix of Alk5 project into the concave cleft of and C). Although both ActRIIA and ActRIIB have been shown to the GDF11 dimer (SI Appendix, Fig. S3 D and E). This results in be capable of signaling with the activin class, other studies have 4 main contact points with GDF11, including the prehelix loop, suggested a preference of GDF8 for ActRIIB (15). Therefore, we fingertip, N terminus, and wrist helix (Fig. 2A and SI Appendix, wanted to further investigate if these observed structural variations Fig. S3A). No major conformational changes (RMSD = 1.76 Å translate into differences in type II receptor binding. over 216 residues, structural alignment) are observed in GDF11 Initially, we tested the ability of the recombinant ECD of both compared with an unbound state (PDB ID code 5E4G), sug- ActRIIA and ActRIIB to inhibit ActA and GDF11 signaling.

2of9 | www.pnas.org/cgi/doi/10.1073/pnas.1906253116 Goebel et al. Downloaded by guest on September 26, 2021 BIOCHEMISTRY

Fig. 2. Differences in receptor specificity for the activin class. (A) Ribbon representation of one half of the GDF11/ActRIIB/Alk5 complex with GDF11/ Alk5 interfaces numbered and labeled accordingly. (B) Inhibition curves (CAGA-luciferase) for respective ligands following the titration of the ECD of ActRIIA (Left) or ActRIIB (Right). (C) Sequence alignment of activin class ligands with fingertip residues represented in red. Numbers correlate to residues in GDF11. Specific molecular interactions between the GDF11 fingertip and the Alk5 β4–β5 loop highlighting the formation of a hydrogen bonding network with dashed lines. (D) Split β-galactosidase dimerization assay (DiscoverX) specific for ActRIIB/Alk5 assembly. For B and D, each data point represents the mean ± SD of triplicate experiments measuring relative luminescence units (RLU) (B)orβ-galactosidase activity (D). For B, 100% signaling represents uninhibited signaling of each respective ligand.

ActRIIB inhibited ActA and GDF11 signaling with similar ments to determine if free receptor could displace ligand-bound IC50 values, whereas ActRIIA inhibited only ActA signaling receptor. Titration of ActRIIA-ECD failed to displace ActRIIB (Fig. 2B). ActRIIA-ECD failed to inhibit GDF11 and GDF8 from a preformed GDF11/ActRIIB complex, whereas ActRIIB signaling (Fig. 2B). Similar to previous studies, mutation of Leu79 could readily displace a GDF11/ActRIIA complex (SI Appendix, in ActRIIB to alanine eliminates ActA binding while maintain- Fig. S5C). In contrast with GDF11, ActRIIA readily displaced ing affinity for GDF11; however, the modified receptor fails to ActRIIB from the ActA/ActRIIB complex (SI Appendix, Fig. inhibit both ActA and GDF11 signaling (SI Appendix, Fig. S4) S5C). Last, equimolar addition of both ActRIIA and ActRIIB to (22). Although direct binding measurements using surface plasmon GDF11 revealed a clear preference for the formation of GDF11/ resonance were complicated as a result of nonspecific interac- ActRIIB binary complex, whereas ActA readily formed a complex tions, native PAGE analysis was used to further analyze the with both receptors (SI Appendix,Fig.S5D). These results suggest interactions, given that binary ligand–receptor complexes are binding differences between activin class ligands and their cognate readily visualized. By titrating increasing amounts of receptors, type II receptors. GDF11 more readily forms a complex with ActRIIB compared The ternary structure also provided insight into type I receptor with ActRIIA, whereas ActA shows little preference for the type specificity. For the activin class, it has been shown that different II receptors (SI Appendix, Fig. S5A). To further test type II re- ligands signal through specific type I receptors, sometimes with ceptor specificity, we purified complexes of ActRIIA and ActRIIB overlapping interactions (16, 17, 23). For example, ActA signals bound to either GDF11 or ActA (SI Appendix,Fig.S5B). Using primarily through Alk4, whereas GDF8 and GDF11 signal these complexes, we performed a series of displacement experi- through both Alk4 and Alk5. Type I receptor specificity has been

Goebel et al. PNAS Latest Articles | 3of9 Downloaded by guest on September 26, 2021 partly attributed to the prehelix loop of the ligand, as ActA can structural comparison highlights significant differences in how be engineered to signal through Alk5 by substitution of the they accommodate this low affinity. Whereas TGFβ utilizes a prehelix loop of GDF8 (24). Examination of the GDF11 ternary cooperative receptor binding mechanism to enhance type I af- receptor complex failed to reveal distinct molecular interactions finity, GDF11 lacks type II and type I receptor interactions and that could account for this observation. However, on the oppo- site end of the type I interface, we noticed significant interactions of the β4–β5 loop of Alk5 with the fingertips of GDF11. Here, a network of hydrogen bonds throughout the fingertip of GDF11 stabilizes interactions with Alk5, specifically Asp81 (Fig. 2C). Interestingly, across the different type I receptors (Alk4, Alk5, and Alk7), the β4–β5 loop exhibits significant variability in length and composition, which, in Alk5, contains a 5-amino acid ex- tension flanked by proline residues that has been shown to be essential for receptor binding (SI Appendix, Fig. S6) (25). Thus, we hypothesized that the fingertip interactions of activin class ligands contribute to type I specificity. Through mutagenesis, we generated both an ActA-GDF8 and an ActA-GDF11 fingertip chimera. Here, we replaced the ActA fingertip residues 405-DDGQN-409 with fingertip residues 354- NGKEQ-358 or 386-NDKQQ-390 of GDF8 and GDF11, re- spectively, with the expectation of conferring Alk5 binding to ActA (Fig. 2C). Purified, recombinant ActA, ActA8Tip,andActA11Tip were generated and shown to signal in a luciferase reporter assay using HEK 293T cells (SI Appendix,Fig.S7). As HEK cells express multiple activin type I receptors, information about receptor spec- ificity cannot be derived. Thus, to determine if the ligand chimeras can interact with Alk5 specifically, we utilized an fragment complementation assay to measure assembly of type I and type II receptors (PathHunter; DiscoverX). ActRIIB and Alk5 were fused to inactive fragments of β-galactosidase and measured for the ability to reconstitute β-galactosidase upon addition of ligand, indicating ligand-mediated receptor dimerization. As expected, titration of GDF8 and GDF11 produced a strong signal, whereas ActA showed no ability to reconstitute β-galactosidase in cells expressing ActRIIB and Alk5 (Fig. 2D). Strikingly, upon titration of ActA11Tip or ActA8Tip, we observed a robust activation of the ActRIIB/Alk5 receptor dimerization assay. These results indicate that ActA can be converted into a ligand that can assemble a signaling complex with Alk5 through specific alteration of fin- gertip residues. These results suggest that type I receptor speci- ficity is derived from the fingertip regions of the ligand, which in turn interact with the β4–β5 loop of the receptor (24). Although similar effects on Alk5 specificity were observed between the mutants, ActA11Tip and ActA8Tip, there were dif- ferences in the potency of Alk5/ActRIIB dimerization whereby GDF11 exhibited a stronger signal than GDF8. This is consistent with previous work in which GDF11 was shown to more potently signal through Alk5 and have a higher apparent binding affinity compared with GDF8, despite only 11 amino acids being different (14, 19). The structure reveals that the majority of the residue differences between GDF8 and GDF11 congregate in the con- Fig. 3. Differential binding of shared type I receptor Alk5 to GDF11 and cave, type I interface (SI Appendix,Fig.S8A). Interestingly, a TGFβ.(A) Ribbon showing 1 type I and 1 type II for GDF11/ActRIIB/Alk5 (PDB β β substitution occurs at the fingertips whereby Asp387 in GDF11 is ID code 6MAC) and TGF 3/T RII/Alk5 (PDB ID code 3KFD), with orientation replaced by Gly355 in GDF8, disrupting the hydrogen bonding consistent with alignment of monomer A of the ligand (9). (B) Surface rep- resentation of GDF11 (Left) and TGFβ3(Right) to highlight the relative po- network of the region (SI Appendix, Fig. S8C). In fact, our pre- 316 sitional differences of monomer B and Alk5. Arrows indicate the directional vious study showed that replacement of either Val of the pre- shift of GDF11 and Alk5 relative to TGFβ3. (C) Overlay of Alk5 bound to 355 helix or Gly of the fingertip in GDF8 with Met or Asp of GDF11 (yellow) and TGFβ3 (sand), respectively. Alignment was performed GDF11, respectively, conferred enhanced signaling activity to using monomer A. Only the receptor is shown to highlight the shifts in both GDF8 (19). These results, together with the structure of the ter- the β4–β5 loop and the N-terminal region. (D) Overlay of GDF11-bound nary complex, indicate that the increase in GDF11 signaling is a Alk5 onto TGFβ3 complex using monomer A for superposition. Circle indi- result of specific changes in the fingertip and prehelix regions that cates a steric clash with the N terminus of the ligand that would occur if β promote more favorable interactions with Alk5. Alk5 were to bind TGF 3 in a similar position as GDF11. (E) Surface inter- actions (within 5 Å) of Alk5 with both GDF11 and TFGβ3, with shared resi- dues in pink and unique ligand-interacting residues in magenta. (F and G) Comparison with the TGFβ Paradigm. The structure of GDF11/ Luciferase reporter assay in R1B L17 cells of GDF11 and TGFβ1 signaling ActRIIB/Alk5 allows us to compare the activin receptor assem- β following transient transfection of 1.25 ng of Alk5 variants and 2.5 ng bly to that of the TGF class and offers a direct comparison of (CAGA)12 promoter (F) or titration of Phe84-Ala (F84A) Alk5 transfection (G) how the 2 ligands engage the same receptor, Alk5 (Fig. 3A). (*P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, 1-way ANOVA). (H) Differences in Although GDF11 and TGFβ both have low affinity for Alk5, the how F84 of Alk5 binds to GDF11 (Top) and TGFβ3(Bottom).

4of9 | www.pnas.org/cgi/doi/10.1073/pnas.1906253116 Goebel et al. Downloaded by guest on September 26, 2021 thus does not use a cooperative mechanism. Instead, GDF11 ligand interface. Most apparent is the placement of Arg82 of utilizes the unique interactions at the fingertip to differentially Alk5, which interacts with the GDF11 fingertips forming a pi interact with Alk5. These differences alter the placement of stack with family-conserved tryptophan residues. For TGFβ3, Alk5 in the type I receptor binding cleft between GDF11 and Arg82 is shifted away from the ligand and contacts the type II TGFβ. Also observed is a significant difference in the configu- receptor, TβRII (SI Appendix, Fig. S9B) (9). Differences also ration of the ligand dimer (Fig. 3B). For TGFβ3, the monomers occur at the C-terminal portion of the β4–β5 loop, where Lys91 of remain in a linear configuration, whereas, for GDF11, monomer Alk5 caps the wrist helix of GDF11 (Fig. 4D). In TGFβ3, this B of the ligand angles upward, moving the N terminus of interaction is missing, and, in fact, residues 90-SKTG-93 of the GDF11 out of the type I binding cleft. The difference in ligand β4–β5 loop are unresolved in the TGFβ3 ternary structure, im- conformation appears necessary for Alk5 to adopt a different plying disorder in this region (SI Appendix, Fig. S9C). position in the type I binding cleft. Superposition of the com- Given these molecular differences, we next sought to de- plexes through monomer A allows one to appreciate the differ- termine the importance of specific Alk5 residues on GDF11 and ences in how Alk5 is oriented relative to the ligand. For GDF11, TGFβ signaling. For this, we used a cell-based luciferase reporter Alk5 positions deeper into the dimer interface, with a shift of 9.2 assay in R1B L17 cells, which lack expression of endogenous Å toward monomer B and a shift of 5.8 Å toward the fingertips C Alk5, and tested the signaling activity of transiently expressed of monomer A (Fig. 3 ). This orientation of Alk5 is restricted wildtype or mutant Alk5. Both wildtype and mutant receptors with TGFβ3 as a result of steric hindrance from the N-terminal were expressed to similar levels (SI Appendix,Fig.S10). As α-helix of the ligand (Fig. 3D). Therefore, GDF11 and TGFβ expected, both GDF11 and TGFβ1 robustly signal through wild- adopt different molecular interactions and assembly mechanisms type Alk5. Conversion of both Asp81-Gly and Arg82-Lys (corre- to bind the same receptor. sponding residues of Alk4) impacted signaling for the ligands, with The differences in Alk5 binding results in both unique and 84 common interactions between GDF11 and TGFβ (Fig. 3E). a larger effect observed for GDF11. Remarkably, Phe -Ala These differences mainly arise from differential interactions with completely abolished GDF11 signaling while maintaining in- β –β β –β teraction with TGFβ1(Fig.3F). These observations were con- the 4 5 loop of Alk5. For GDF11, the 4 5 loop contacts the 84 ligand fingertip, which in turn curls around the receptor. This is firmed through titration of wildtype and Phe -Ala Alk5, in which β in contrast to the flatter extended conformation of the TGFβ3 TGF 1 signaling remains consistent, whereas GDF11 signals fingers, which interact with the β4–β5 loop using a combination through only wildtype Alk5 (Fig. 3G). Comparison of the com- 84 BIOCHEMISTRY of the ligand fingers and the N terminus of TβRII (9). Inter- plexes reveals differences in how Phe is positioned relative to estingly, the curved fingertip of GDF11 seems to parallel the GDF11 and TGFβ. In the GDF11 complex, Phe84 forms a “knob- interreceptor interactions between TβRII and Alk5 (Fig. 2C and in-hole” interaction with a hydrophobic cleft facilitated by the SI Appendix, Fig. S9B). Thus, even though GDF11 does not family-conserved tryptophan residues, whereas, in complex with utilize interreceptor interactions to facilitate Alk5 binding, the TGFβ,Phe84 is shifted, forming a tangential interaction with the curvature of the fingertips provides a similar framework. Further hydrophobic surface of the ligand (Fig. 3H). This support the molecular differences are observed across the type I receptor– conclusion that, even though both GDF11 and TGFβ signal using

Fig. 4. Comparison of type I receptor engagement across the TGFβ family. (A) Ribbon showing 1 type I and 1 type II for GDF11/ActRIIB/Alk5 (PDB ID code 6MAC) and BMP2/ActRIIA/Alk3 (PDB ID code 2GOO), with orientation consistent with alignment of monomer A of the ligand. (B) Knob-in-hole motif observed between F108 of Alk3 and BMP2. (C and D) Comparison of the ligand fingertip-type I interface (C) and the ligand wrist helix cap (D) between GDF11/Alk5 (Left) and BMP2/Alk3 (Right). (E) Ligand surface representation of type I binding interface, with interacting residues on monomer A colored magenta and those on monomer B colored pink. Buried surface area calculations for each of the interfaces are shown as determined by using jsPISA.

Goebel et al. PNAS Latest Articles | 5of9 Downloaded by guest on September 26, 2021 the same type I receptor, the ligands have adopted different strategies affinity binding and antagonism to both GDF8 and GDF11 (27, to accommodate low-affinity interactions with Alk5. 28). From previous structures of ligand–receptor complexes, the general binding interface of the type II receptor for GDF11 was Comparison with the BMP Paradigm. At first glance, the structure anticipated; however, the structure of ternary GDF11 complex of GDF11/ActRIIB/Alk5 mimics the receptor orientation ob- highlights ligand differences within the activin class that account served with the BMP2 and BMP9 receptor assemblies, where the for preferential interactions with type II receptors. Using only type I receptor binds independently from the type II receptor the ECD of the type II receptors, we further investigated the (Fig. 4A) (7, 8, 11). In addition, there are similar molecular fea- preference of GDF11 and ActA for type II receptors. We show tures throughout the interface that indicate common mechanisms that ActRIIB, and not ActRIIA, can inhibit GDF11 signaling in of binding. Specifically, Alk3 and Alk5 utilize a knob-in-hole a luciferase reporter assay. Native gel analysis shows a similar strategy whereby a phenylalanine (Phe108-Alk3 and Phe84-Alk5) preference of GDF11 for ActRIIB over ActRIIA. This prefer- projects into a hydrophobic pocket in the ligand (Figs. 3H and 4B) ence is not observed with ActA, in which complex formation is (7). Similarly, Arg82 of Alk5 and Lys115 in Alk3 perform a similar similar between ActRIIA and ActRIIB. Interestingly, in the role by forming a hydrophobic interaction with the family-conserved presence of equal quantities of ActRIIA and ActRIIB, GDF11 tryptophan residues of the ligand (Fig. 4C). Last, both Alk3 and forms complex with only the latter, suggesting that GDF11 likely Alk5 utilize the C-terminal end of the β4–β5looptocapthewrist prefers ActRIIB when both receptors are present at the cellular helix of the ligand (Fig. 4D)(7). surface. This preference is not observed for ActA, in which the li- Despite these similarities, the structure highlights major dif- gand can bind to either type II receptor. For GDF11, this might ferences between the GDF11 and BMP complexes in how the ensure that only 1 type II receptor binds in the ternary complex, type I receptor binds at ligand dimer interface. Here, the BMP whereas ActA has the potential to form a ternary complex that complex utilizes a much larger degree of surface area to bind the contains 1 ActRIIA and 1 ActRIIB. This implies that cell surface type I receptor compared with the GDF11 complex. This is representation of the type II receptors, which are different in several consistent with the relative affinities for the type I receptors, in tissues, might be used to modulate ligand sensitivity (22, 29, 30). which, in general, BMP ligands bind with higher affinity than Given the similarities in general receptor positioning between activin class ligands. Specifically, interaction of BMP2 with GDF11 and BMP ligands, it is interesting to consider why ligands Alk3 buries 1,350 Å2 versus 940 Å2 for the GDF11 Alk5 in- have developed different strategies for binding type I receptors. terface (8). This difference is a result of more substantial inter- A major difference in the ligands appears in the flexibility of the actions between the receptor and monomer B of BMP2, which dimer interface. For the activin class, multiple ligands have been buries an additional 480 Å2 over the monomer B of GDF11 (Fig. shown to exhibit significant flexibility of the dimer interface, 4E). This increase in surface area is a result of multiple residues which disrupts the architecture of the type I binding cleft. This is within the prehelix loop of BMP2 extending into the palm of supported by various structures, including the recent structures Alk3 compared with GDF11, which interacts with Alk5 through of GDF11 and GDF8, in which the ligand is described in an only a single residue (Met348). However, in contrast, GDF11 “open” (fully formed type I cleft) or a “closed” state typically buries slightly more surface area on monomer A as a result of associated with a disordered wrist region (13, 19, 20, 24, 31–37). increased interactions between the β4–β5 loop of Alk5 and the In contrast, BMP ligands are consistently observed in the open fingertips. Interestingly, BMP type I receptors Alk3 and Alk6 ligand state, implying rigidity of the ligand dimer. It is possible have shorter β4–β5 loops, which limits the ability to interact with that the flexibility of activin class ligands prevents interactions the fingertip of the ligand (Fig. 4C and SI Appendix, Fig. S6). with BMP type I receptors, such as Alk3 and Alk6, which may Thus, fingertip interactions are unique to the GDF11 receptor favor a more rigid type I interface. Given the evidence, it is in- assembly, further differentiating the activin paradigm of receptor triguing to speculate that activin ligand flexibility might be used assembly from that of TGFβ and BMP. Although the apparent to accommodate the positioning of type I receptors and that, assembly mechanism appears similar between the BMP and along with low affinity, might allow certain ligands to be more activin class, differences in affinity and surface area utilization promiscuous with type I receptors. lead to diverging receptor assembly mechanisms. Previous studies have implicated the prehelix loop as an ele- ment for determining type I receptor specificity (24). For BMP Conclusions. Studies within the TGFβ family have revealed dis- ligands, specific interactions of the prehelix loop are important tinct mechanisms for receptor assembly that are defined by re- for type I interactions. For BMP2, single changes in the prehelix ceptor affinities, receptor positioning, and SMAD activation: the loop can render the ligand deficient in receptor binding (e.g., TGFβ paradigm, which utilizes cooperative receptor interactions L51P), whereas activin ligands are less dependent on specific during assembly, and the BMP paradigm, which utilizes a non- residues (6, 38). In line with this, specific differences within the cooperative lock-and-key receptor binding mode (7, 9, 14). A prehelix loop between BMP2 and GDF5 have been shown to third class, the activins, has commonalities with both paradigms: directly account for differences in utilization of type I receptors receptor affinities similar to that of the TGFβ class and receptor Alk3 and Alk6 (39–41). Although significant variations of the orientation similar to that of the BMP class. However, how the prehelix loop exist across activin class members (SI Appendix, activins engage and interact with their cognate type I receptors to Fig. S6), it is possible that these differences account for differ- form specific contacts remains uncharacterized. The structure of ences in ligand flexibility, which might impact how particular li- GDF11/ActRIIB/Alk5 provides insights into the activin class and gands accommodate type I receptors. context for type I and type II interactions across the broader The study identified that additional elements of type I speci- TGFβ family. ficity occur in the fingertip region of the ligand. We show that Previous biochemical work has suggested that activin class li- GDF11 uses the fingertip region to engage the β4–β5 loop of gands interact differently with the type II receptors ActRIIA and Alk5, forming a network of hydrogen bonds. The variation in ActRIIB (16, 23). However, for GDF11, both ActRIIA and fingertip composition observed in both GDF8 and ActA likely ActRIIB have been shown to be important for signaling during weakens this network, therefore destabilizing Alk5 interactions. the patterning of axial vertebrae, implying an ability to signal Structural comparison across the TGFβ family reveals that the using either type II receptor (26). Previous crosslinking and interactions between the fingertip and the β4–β5 loop are unique to binding studies have indicated that GDF8 has a preference for the activin class (Fig. 4C and SI Appendix,Fig.S9B). Interestingly, ActRIIB (15, 16). In contrast, studies using the Fc-fusion forms activin type I receptors Alk4 and Alk5 uniquely possess an exten- of ActRIIA or ActRIIB, which benefit from avidity, show high- sion in the β4–β5 loop, allowing them to extend interactions into

6of9 | www.pnas.org/cgi/doi/10.1073/pnas.1906253116 Goebel et al. Downloaded by guest on September 26, 2021 the fingertip interface, indicating a divergence of type I receptors for interactions with activin class ligands. Interestingly, this extension is absent in Alk7 and might provide differential interactions with activin class ligands. Therefore, our study identifies the fingertip as a major determinant of type I specificity for the activin class. Comparison of Alk5 with the BMP receptor Alk3 revealed a common knob-in-hole motif. Here, a phenylalanine wedges into a hydrophobic pocket created at the dimer interface. Sequence alignment of the type I receptors reveals a phenylalanine or equivalent hydrophobic residue in similar positions in all type I receptors, save for Alk1, which uses a glutamate residue to form hydrogen bonds with the ligand (11). For Alk5, this interaction is not observed in complexes with TGFβ in which the phenylala- nine adopts a different sidechain rotamer and does not protrude into a hydrophobic cleft. Remarkably, mutation of Phe84 to al- anine abolished GDF11 signaling through Alk5 but did not dis- rupt TGFβ signaling, indicating that the 2 ligands differently utilize receptor interactions. This indicates that TGFβ is less sensitive than GDF11 to disruptive mutations at the type I ligand interface, presumably because of the additional contacts offered by the type II receptor, TβRII. Given that full signaling complexes of each class have been Fig. 5. Receptor assembly paradigms of the TGFβ superfamily. (A) Full sig- resolved structurally, it is interesting to compare overall receptor naling complex structures from across the TGFβ superfamily: activin (PDB ID positioning across the family (Fig. 5A). At a glance, both BMP code 6MAC), TGFβ (PDB ID code 3KFD), and BMP (PDB ID code 2GOO) (8, 9). and activin ligands position receptors in a similar manner, with Type II and type I receptors are represented in orange and yellow, re- TGFβ shifting the type II binding interface toward the fingertip, spectively, with mature ligands in blue. (B) Cartoon representation of the receptor surface complex with cis- and trans-receptor pairs labeled. (C) Dis-

allowing direct interreceptor interactions to occur with the same- BIOCHEMISTRY B tances are calculated from the center of mass for each receptor for cis- and sided (cis-) type I receptor (Fig. 5 ). This cis-receptor pair alone trans-receptor combinations. (D) Schematic representation depicting half has been shown, through mutagenesis, to be sufficient for TGFβ the signaling complex for the 3 receptor binding mechanisms of TGFβ family signaling (42). For BMP ligands, 2 type II receptors are required, ligands. whereas only 1 type I receptor is sufficient for signaling (43). Whether similar signaling pairs are sufficient for activin class signaling has yet to be determined. However, the GDF11 ternary Materials and Methods complex indicates that the overall receptor spacing is similar for ActRIIB, ActRIIA, and Alk5 Expression and Purification. The extracellular do- the BMP and activin class ligands, even though, for GDF11, the mains of rat ActRIIB (residues 1–120) and human ActRIIA (residues 1–134) type I receptor interacts more extensively with the ligand fin- were subcloned into the pVL1392 baculovirus vector. ActRIIB was cloned with a C-terminal thrombin cleavage site and a His tag, whereas ActRIIA gertips (Fig. 5C). 6 was cloned with C-terminal Flag and His10 tags. Recombinant baculoviruses In this study, we show that the 3 classes of ligands utilize were generated by using the Baculogold system (ActRIIB; Pharmingen) or different molecular strategies to bind type I receptors. TGFβ the Bac-to-Bac system (ActRIIA; Invitrogen). Virus amplification and protein utilizes a cooperative mechanism between type I and type II expression were carried out using standard protocols in SF+ insect cells receptors, whereas the BMP class uses a high-affinity lock-and- (Protein Sciences). ActRIIB and ActRIIA were purified from cell supernatants key mechanism (Fig. 5D) (8, 9). The activin class sets itself apart by using Ni Sepharose affinity resin (GE Healthcare) with buffers containing from the TGFβ and BMP classes in which type I receptor 50 mM Na2HPO4, pH 7.5, 500 mM NaCl, and 20 mM imidazole for loading/ specificity is more promiscuous (5, 15–18). This promiscuity is washing and 500 mM imidazole for elution. ActRIIB was then digested with apparent for ActA, which, in addition to activating SMAD2/ thrombin overnight to remove the His6 tag. ActRIIA and ActRIIB were sub- jected to size exclusion chromatography (SEC) using a HiLoad Superdex S75 3 through Alk4, can form a nonsignaling complex with BMP type 16/60 column (GE Healthcare) in 20 mM Hepes, pH 7.5, and 500 mM NaCl. I receptor Alk2, acting as a competitive inhibitor. However, a Additionally, human Alk5 (residues 1–101) was expressed in Escherichia coli single intracellular point mutation of Alk2 switches ActA to an and was prepared through an oxidative refolding/HPLC purification pro- agonist, leading to aberrant ossification and the disease fibro- cedure described previously (44). dysplasia ossificans progressiva. Taken together, our structural and specificity data support a third mechanism of ligand–re- Mature ActA, GDF11, GDF8, and TGFβ1 Expression and Purification. The DNA ceptor interactions whereby subtle differences in the ligand im- sequence encoding for human ActA and human GDF11 was cloned into the pact the binding of low-affinity type I receptors, as observed with pAID4T vector containing a UCOE element (Millipore) for increased expression. The plasmids were stably transfected in Chinese hamster ovary the fingertip (Fig. 5D). This is consistent with a mechanism (CHO) DUKX cells, and pools were generated and used to express ActA and whereby the high-affinity type II receptor localizes the ligand on GDF11. Conditioned media (CM) containing ActA was mixed with a pro- the cell surface, concentrating the ligand to facilitate interactions prietary affinity resin made with an ActRIIA-related construct (Acceleron). with low-affinity type I receptors (34). We hypothesize that li- The pH of the solution was lowered to 3.0 to dissociate the propeptide–ligand gand flexibility is used to maintain low-affinity type I interactions complex and raised to 7.5 after a few minutes, and the solution was in- and increase the type I receptor binding profile by facilitating a cubated for 2 h at room temperature. The resin was filtered to remove the conformational selection model. The reason the activin class has conditioned media and then washed with PBS solution and eluted with evolved this mechanism and not simply a different lock-and-key 0.1 M glycine, pH 3.0. The glycine elution was concentrated over a phenyl fit remains unclear, but suggests that type I receptor promiscuity hydrophobic interaction column (GE Healthcare) and eluted with 50% ace- tonitrile/water with 0.1% trifluoroacetic acid (TFA) and then further con- might play an important, yet unknown, biological role. Ulti- centrated by centrifugation in Amicon tubes. The protein was then purified mately, additional structural data are needed to compare and over a preparative reverse-phase C4 column (Vydac) attached to an HPLC contrast receptor specificity within the activin class of TGFβ with a gradient of water/0.1% TFA and acetonitrile/0.1% TFA. Fractions signaling ligands. containing pure protein were combined and lyophilized. The CM containing

Goebel et al. PNAS Latest Articles | 7of9 Downloaded by guest on September 26, 2021 GDF11 was filtered, concentrated, heated to 75 °C, and cooled to room lattice was determined through molecular replacement using Phaser and the temperature before protein purification. GDF11 was purified by using a search models ActRIIB (PDB ID code 1NYS), GDF11 (PDB ID code 5E4G), and proprietary affinity resin based on an ActRIIB-related construct. The resin Alk5 (PDB ID code 3KFD) (9, 13, 20, 49). Final refinement and model building was incubated with the prepared CM for 2 h with shaking, followed by a were performed with Phenix.Refine, PDB-REDO, and Coot (SI Appendix, wash with PBS solution and elution with 0.1 M glycine, pH 3.0. Purified Table S1)(50–52). Final coordinates are available in the PDB under ID code material was applied to a preparative C4 reverse-phase column and purified 6MAC. All native PAGE analysis was carried out as described previously (53). as described with ActA. Pure protein fractions were pooled and dialyzed in To determine the composition from native gels, particular bands were ex- 10 mM HCl and then dried with a refrigerated speed vacuum concentrator cised and electro-eluted followed by analysis on SDS/PAGE. at −4 °C. For both proteins, purity was assessed by SDS/PAGE analysis. Ad-

ditionally, mature, human GDF8 was purchased through R&D. Human Luciferase Reporter Assays. Assays using the HEK-293-(CAGA)12 luciferase TGFβ1 was expressed and purified from conditioned medium produced by reporter cells (initially derived from RRID: CVCL_0045) were performed as an overexpressing stably transfected CHO line described previously (45). previously described (19, 24). Briefly, cells were plated in 96-well format (3 × 104 cells per well) and grown for 24 h. For the inhibition comparison assays ActA Propeptide and ActA Fingertip Mutant Expression and Purification. ActA featured in Fig. 2, the growth media was removed and replaced with serum- constructs were untagged and cloned downstream of mouse ActA propep- free media containing 0.1% BSA and a 2-fold serial titration series of tide (with native signal sequence) by isothermal Gibson assembly of synthetic ActRIIA, ActRIIB, or ActRIIB:L79A ECD (0.25 to 160 nM) with constant ligand gene blocks (Integrated DNA Technologies) into an expression vector driven (ActA, GDF11, GDF8) concentration (0.62 nM). Cells were lysed, and lumi- by CMV promoter (46). For purification, propeptide complexes were secreted nescence was measured 18 h after treatment by using a Synergy H1 Hybrid from CHO-K1 cells generated by Regeneron Protein Expression Sciences plate reader (BioTek). For assays utilizing the R1B L17 cell line (RRID: Group using proprietary EESYR technology. Noncovalently bound mature CVCL_0596), cells were seeded into the 96-well format (3 × 104 cells per well) ActA dimer in complex with ActA propeptide was purified to homogeneity and grown for 24 h. The cells were then cotransfected with a total of 100 ng

by heparin chromatography, followed by SEC at 4 °C, and the same purifica- DNA containing the (CAGA)12 luciferase reporter construct and wildtype or tion protocol was used for both wild type Activin A and fingertip mutant mutant receptor containing plasmids (pRK5 rat Alk5 S278T [ST] variant) using complexes. Specifically, CHO-K1 conditioned media was applied without Mirus LT-1 transfection reagent. The ST variant is not inhibited by SB-431542, modification to 2 tandem HiTrap Heparin columns (GE Healthcare/Pharmacia) reducing background signaling from low-level basal type I expression (54). that were equilibrated in 20 mM Tris·HCl, pH 7.4, 0.15 M NaCl. Subsequent to Empty pRK5 vector was added to normalize the total DNA concentration. sample loading, heparin columns were washed with 15 column volumes of Following overnight transfection, media was exchanged to serum-free media 20 mM Tris·HCl, 0.15 M NaCl, pH 7.4, and then 15 column volumes of 20 mM containing 0.1% BSA, 0.62 nM (or titration of) mature GDF11 or TGFβ1, and Tris·HCl, 0.3 M NaCl, pH 7.4. Bound Activin A propeptide complexes were 10 uM SB-431542. Cells were lysed, and luminescence was measured 18 h after eluted with a linear NaCl gradient from 0.3 to 1.0 M. Pooled fractions were ligand treatment. concentrated and applied to a Sephadex S200 HiPrep column (GE Healthcare/ Pharmacia) equilibrated with 20 mM Tris·HCl, pH 7.4, and 0.3 M NaCl. PathHunter Dimerization Assays. The PathHunter eXpress ActRIIB/Alk5 di- merization assays (93-1046E3) were developed by DiscoverX. In this assay, Crystallization and Structure Determination. Complex formation between human ActRIIB (residues 1–164) and human Alk5 (residues 1–158) are C- recombinant GDF11 and ActRIIB was performed by mixing the 2 proteins at a terminally truncated to facilitate fusion to fragments of the β-galactosidase 2.1:1 molar ratio, followed by incubation at room temperature for 1 h and (β-gal) enzyme. Specifically, ActRIIB is fused to a small fragment of β-gal called SEC in 20 mM Hepes, pH 7.5, and 500 mM NaCl. A complex peak was observed the enzyme donor, PK1; Alk5 is fused to a larger fragment called the enzyme and contained both GDF11 and ActRIIB based on SDS/PAGE analysis. The same acceptor, EA; and both receptors are coexpressed within a human osteosar- process was utilized for each binary complex formed for the native PAGE coma cell line (U2OS). During receptor assembly with a cognate ligand, the analysis featured in SI Appendix, Fig. S5. To establish appropriate mixing fragments come together and form active β-galactosidase enzyme that hy- ratios of GDF11/ActRIIB binary complex and Alk5 for crystallization, a titra- drolyzes substrate to generate a chemiluminescent signal. Cells from the U2OS tion of Alk5 was performed through native PAGE analysis and optimized to clones were plated in a 96-well format (1 × 104 cells per well) and grown for 1:2.1 to minimize the amount of free binary or Alk5. The complex was 24 h in Eagle’s minimum essential medium supplemented with 2% FBS and 1% concentrated to 8 mg/mL and set up by using the hanging drop method in penicillin/streptomycin. Media was then exchanged to serum-free media con- crystallization trials. The final ternary complex crystal was grown in mother taining 0.1% BSA and a titration of ligand (∼1pM–80 nM). Following treat- liquor containing 0.1 M sodium thiocyanate, 0.1 M sodium acetate, and 16% ment for 16 h, samples were treated with PathHunter FLASH substrate and polyethylene glycol 8000 at pH 6 and 20 °C. Mother liquor supplemented detection reagents (DiscoverX) followed by chemiluminescence measurement with 25% ethylene glycol was used as a cryoprotectant for crystal freezing. according to the manufacturer’sprotocol. X-ray diffraction data were collected at GM-CA beamline 23ID-B of the Advanced Photon Source at Argonne National Laboratory. Data were ACKNOWLEDGMENTS. The authors thank Drs. Michael Becker and Qingping indexed and integrated with DIALS and subsequently scaled and merged Xu and the staff of the 23-ID Beamline at the Advanced Photon Source, through AIMLESS (47, 48). Placement of the ternary complex in the crystal Argonne National Laboratory, for assistance in X-ray data collection.

1. A. P. Hinck, T. D. Mueller, T. A. Springer, Structural biology and evolution of the TGF- b 12. J. Greenwald et al., The BMP7/ActRII extracellular domain complex provides new in- family. Cold Spring Harb. Perspect. Biol. 8, a022103 (2016). sights into the cooperative nature of receptor assembly. Mol. Cell 11, 605–617 (2003). 2. A. P. Hinck, Structural studies of the TGF-betas and their receptors–Insights into 13. T. B. Thompson, T. K. Woodruff, T. S. Jardetzky, Structures of an ActRIIB:activin A evolution of the TGF-beta superfamily. FEBS Lett. 586, 1860–1870 (2012). complex reveal a novel binding mode for TGF-beta ligand:receptor interactions. 3. A. Weiss, L. Attisano, The TGFbeta superfamily signaling pathway. Wiley Interdiscip. EMBO J. 22, 1555–1566 (2003). Rev. Dev. Biol. 2,47–63 (2013). 14. D. Yadin, T. D. Mueller, Structural insights into BMP receptors: Specificity, activation 4. L. Attisano et al., Identification of human activin and TGFb type I receptors that form and inhibition. Growth Factor Rev. 27,13–34 (2016). heteromeric kinase complexes with type II receptors. Cell 75, 671–680 (1993). 15. S.-J. Lee, A. C. McPherron, Regulation of activity and muscle growth. Proc. 5. J. L. Wrana, L. Attisano, R. Wieser, F. Ventura, J. Massague, Mechanism of activation Natl. Acad. Sci. U.S.A. 98, 9306–9311 (2001). of the TGF-β receptor. Nature 370, 341–347 (1994). 16. A. Rebbapragada, H. Benchabane, J. L. Wrana, A. J. Celeste, L. Attisano, Myostatin 6. S. Keller, J. Nickel, J.-L. Zhang, W. Sebald, T. D. Mueller, Molecular recognition of signals through a transforming growth factor beta-like signaling pathway to block BMP-2 and BMP receptor IA. Nat. Struct. Mol. Biol. 11, 481–488 (2004). adipogenesis. Mol. Cell. Biol. 23, 7230–7242 (2003). 7. D. Weber et al., A silent H-bond can be mutationally activated for high-affinity in- 17. O. Andersson, E. Reissmann, C. F. Ibáñez, Growth differentiation factor 11 signals teraction of BMP-2 and activin type IIB receptor. BMC Struct. Biol. 7, 6 (2007). through the transforming growth factor-beta receptor ALK5 to regionalize the 8. G. P. Allendorph, W. W. Vale, S. Choe, Structure of the ternary signaling complex of a anterior-posterior axis. EMBO Rep. 7, 831–837 (2006). TGF-beta superfamily member. Proc. Natl. Acad. Sci. U.S.A. 103, 7643–7648 (2006). 18. K. Tsuchida, M. Nakatani, A. Uezumi, T. Murakami, X. Cui, 9. J. Groppe et al., Cooperative assembly of TGF-β superfamily signaling complexes is pathway through activin receptors as a therapeutic target of musculoskeletal diseases mediated by two disparate mechanisms and distinct modes of receptor binding. Mol. and cancer. Endocr. J. 55,11–21 (2008). Cell 29, 157–168 (2008). 19. R. G. Walker et al., Structural basis for potency differences between GDF8 and GDF11. 10. S. Radaev et al., Ternary complex of transforming growth factor-β1 reveals isoform- BMC Biol. 15, 19 (2017). specific ligand recognition and receptor recruitment in the superfamily. J. Biol. Chem. 20. A. K. Padyana et al., Crystal structure of human GDF11. Acta Crystallogr. F Struct. Biol. 285, 14806–14814 (2010). Commun. 72, 160–164 (2016). 11. S. A. Townson et al., Specificity and structure of a high affinity activin receptor-like 21. E. Krissinel, Stock-based detection of protein oligomeric states in jsPISA. Nucleic Acids kinase 1 (ALK1) signaling complex. J. Biol. Chem. 287, 27313–27325 (2012). Res. 43, W314–W319 (2015).

8of9 | www.pnas.org/cgi/doi/10.1073/pnas.1906253116 Goebel et al. Downloaded by guest on September 26, 2021 22. D. Sako et al., Characterization of the ligand binding functionality of the extracellular 38. K. Heinecke et al., Receptor oligomerization and beyond: A case study in bone domain of activin receptor type IIB. J. Biol. Chem. 285, 21037–21048 (2010). morphogenetic proteins. BMC Biol. 7, 59 (2009). 23. S.-J. J. Lee, Regulation of muscle mass by myostatin. Annu. Rev. Cell Dev. Biol. 20,61– 39. U. Klammert et al., GDF-5 can act as a context-dependent BMP-2 antagonist. BMC 86 (2004). Biol. 13,1–18 (2015). 24. J. N. Cash, C. A. Rejon, A. C. McPherron, D. J. Bernard, T. B. Thompson, The structure of 40. J. Nickel, A. Kotzsch, W. Sebald, T. D. Mueller, A single residue of GDF-5 defines myostatin: 288: Insights into receptor utilization and heparin binding. binding specificity to BMP receptor IB. J. Mol. Biol. 349, 933–947 (2005). EMBO J. 28, 2662–2676 (2009). 41. P. Seemann et al., Activating and deactivating mutations in the receptor interaction 25. J. E. Zuniga et al., The TβR-I pre-helix extension is structurally ordered in the unbound site of GDF5 cause symphalangism or brachydactyly type A2. J. Clin. Invest. 115, 2373– form and its flanking prolines are essential for binding. J. Mol. Biol. 412, 601–618 2381 (2005). (2011). 42. T. Huang et al., TGF-β signalling is mediated by two autonomously functioning 26. S. Paul Oh et al., Activin type IIA and IIB receptors mediate Gdf11 signaling in axial TβRI:TβRII pairs. EMBO J. 30, 1263–1276 (2011). vertebral patterning. Dev. 16, 2749–2754 (2002). 43. P. Knaus, W. Sebald, of binding epitopes and receptor chains in the 27. S. Aykul, E. Martinez-Hackert, Transforming growth factor-β family ligands can BMP/TGFβ superfamily. Biol. Chem. 382, 1189–1195 (2001). function as antagonists by competing for type II receptor binding. J. Biol. Chem. 291, 44. J. E. Zúñiga et al., Assembly of TβRI:TβRII:TGFβ ternary complex in vitro with receptor 10792–10804 (2016). extracellular domains is cooperative and isoform-dependent. J. Mol. Biol. 354, 1052– 28. M. Dussiot et al., An activin receptor IIA ligand trap corrects ineffective erythropoiesis 1068 (2005). in β-thalassemia. Nat. Med. 20, 398–407 (2014). 45. Z. Zou, P. D. Sun, Overexpression of human transforming growth factor-β1 using a 29. B. C. Goh et al., Activin receptor type 2A (ACVR2A) functions directly in osteoblasts as recombinant CHO cell expression system. Protein Expr. Purif. 37, 265–272 (2004). a negative regulator of bone mass. J. Biol. Chem. 292, 13809–13822 (2017). 46. D. G. Gibson et al., Supporting online material for creation of a bacterial cell con- 30. O. E. Olsen et al., Activin A inhibits BMP-signaling by binding ACVR2A and ACVR2B. trolled by a chemically synthesized genome. Science 329,1–6 (2010). Cell Commun. Signal. 13,1–7 (2015). 47. P. R. Evans, G. N. Murshudov, How good are my data and what is the resolution? Acta 31. J. N. Cash et al., Structure of myostatin·follistatin-like 3: N-terminal domains of Crystallogr. Sect. D. Biol. Crystallogr. 69, 1204–1214 (2013). follistatin-type molecules exhibit alternate modes of binding. J. Biol. Chem. 287, 48. M. T. B. Clabbers, T. Gruene, J. M. Parkhurst, J. P. Abrahams, D. G. Waterman, Electron 1043–1053 (2012). diffraction data processing with DIALS. Acta Crystallogr. Sect. D. Struct. Biol. 74, 506– 32. A. E. Harrington et al., Structural basis for the inhibition of activin signalling by 518 (2018). follistatin. EMBO J. 25, 1035–1045 (2006). 49. A. J. Mccoy et al., Phaser crystallographic software. J. Appl. Cryst. 40, 658–674 (2007). 33. B. H. Yoon et al., An activin A/BMP2 chimera, AB204, displays bone-healing properties 50. P. V. Afonine, W. Ralf, J. J. Headd, C. Thomas, Towards automated crystallographic superior to those of BMP2. J. Bone Miner. Res. 29, 1950–1959 (2014). structure refinement with phenix refine. Acta Cryst. D68, 352–367 (2012). 34. J. Greenwald et al., A flexible activin explains the membrane-dependent cooperative 51. R. P. Joosten, F. Long, G. N. Murshudov, A. Perrakis, The PDB_REDO server for mac- assembly of TGF-β family receptors. Mol. Cell 15, 485–489 (2004). romolecular structure model optimization. IUCrJ 1, 213–220 (2014). 35. R. Stamler et al., The structure of FSTL3.activin A complex: Differential binding of N- 52. P. Emsley, B. Lohkamp, W. G. Scott, K. Cowtan, Features and development of Coot. terminal domains influences follistatin-type antagonist specificity. J. Biol. Chem. 283, Acta Crystallogr D. Biol Crystallogr. 66, 486–501 (2010). 32831–32838 (2008). 53. R. G. Walker et al., Molecular characterization of latent GDF8 reveals mechanisms of 36. X. Wang, G. Fischer, M. Hyvönen, Structure and activation of pro-activin A. Nat. activation. Proc. Natl. Acad. Sci. U.S.A. 115, E866–E875 (2018).

Commun. 7, 12052 (2016). 54. G. J. Inman, SB-431542 is a potent and specific inhibitor of transforming growth BIOCHEMISTRY 37. T. R. Cotton et al., Structure of the human myostatin precursor and determinants of factor-beta superfamily type I activin receptor-like kinase (ALK) receptors ALK4, growth factor latency. EMBO J. 37, 367–383 (2018). ALK5, and ALK7. Mol. Pharmacol. 62,65–74 (2002).

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