Volume 11, Issue 3, Pages 605-617 (March 2003) The BMP7/ActRII Extracellular Domain Complex Provides New Insights into the Cooperative Nature of Receptor Assembly  Jason Greenwald, Jay Groppe, Peter Gray, Ezra Wiater, Witek Kwiatkowski, Wylie Vale, Senyon Choe  Molecular Cell  Volume 11, Issue 3, Pages 605-617 (March 2003) DOI: 10.1016/S1097-2765(03)00094-7 Copyright © 2003 Cell Press Terms and Conditions

Figure 1 The Structure of the BMP7/ActRII-ECD Complex (A and B) Ribbon diagrams of the BMP7/ActRII-ECD complex (A) with the 2-fold symmetry axis vertical and the membrane facing side at the bottom and (B) the view from above (BMP7, gold and rust; ActRII-ECD, green; cystine sulfurs, yellow space-filling). (C) Stereo view of the interface between BMP7 and ActRII in an orientation close to (A). The residues within 4 Å of the binding partner as well as Glu29 are displayed as balls and sticks. Glu29 and those residues whose mutations are known to affect binding (pink, Figure 2) are labeled. In (A)–(C) significant conformational changes are highlighted (dark blue), and in (C) they are overlaid with the unbound conformations (light blue). This figure was made using MOLSCRIPT (Kraulis, 1991). Molecular Cell 2003 11, 605-617DOI: (10.1016/S1097-2765(03)00094-7) Copyright © 2003 Cell Press Terms and Conditions

Figure 2 Sequence Alignments of TGF-β Superfamily Ligands and Type II Receptor ECDs (A and B) Residue numbers above the sequences refer to ActRII and BMP7 and secondary structure elements refer to the complex. Residues that come within 4 Å of the binding partner at the type II interfaces are boxed and shaded green or pink, and those at the type I interface (BMP2) are boxed. The individual residues of activin, BMP2, and ActRII that have been shown by mutagenesis to be important for type II receptor binding are shaded in pink. The cysteines are shaded yellow and the conserved cysteines of the folds are boxed. The regions of conformational change (A and B loops) are lettered in blue. The alignments were performed by CLUSTALW (Thompson et al., 1994) using profiles from a structural alignment caclulated by STAMP (Russell and Barton, 1992). The gray boxes indicate the regions defined by the STAMP alignment procedure. The cysteine score in the Gonnet matrix was modified to ensure correct alignment of the conserved cysteines. (A) and (B) were made using ALSCRIPT (Barton, 1993). (C) A space-filling model of the complex dissociated by a 180° rotation of ActRII from the right half of the complex in Figure 1A. The colors are as in (A) and (B) with the BMP2 mutations mapped onto BMP7. (D) Catalog of BMP7-ActRII contacts showing the types of interactions and the conservation of each residue. The residue colors are as in (C) and their conservation (BMP7 with activin and ActRII with BMPRII) is shown as black bars for identical residues, gray for conservative changes, and white for nonconservative changes. The interaction type is colored yellow for nonpolar-nonpolar, blue for h-bonded, and white for nonpolar-polar. *, the conserved hydrophobic core residues of the interface; –, main chain-side chain contacts; all others are side chain-side chain. Molecular Cell 2003 11, 605-617DOI: (10.1016/S1097-2765(03)00094-7) Copyright © 2003 Cell Press Terms and Conditions

Figure 3 The Model of the BMP7/ActRII/BMPRIa Six-Chain Signaling Complex BMPRIa (purple) was placed in the complex by aligning the BMP2/BMPRIa structure (Kirsch et al., 2000a) with BMP7. (A) Side view as in Figure 1A is shown as a solvent accessible surface. The horizontal line represents the plane of the membrane. (B) Bottom view (opposite from Figure 1B). Sugars are in black. The C termini are marked with yellow dots and the horizontal distances between them as projected onto the plane of the membrane are 83 Å for type II-type II, 66 Å for type I-type I, and 27 Å and 68 Å for type I-type II. (A) and (B) were prepared with DINO (Philippsen, 2001). (C) Stereo view of ActRII and TGF-βRII bound to their respective ligands overlaid with the BMP2/BMPRIa structure. The BMP7/ActRII complex was aligned as in (A) using the entire ligand. In order to overlay the TGF-βRII binding site (tip of finger 2 on TGF-β3) with BMP2, only 14 residues (86–92 and 98–104 of BMP2) were used for the alignment (Hart et al., 2002). The color scheme is BMP2, white; BMP7, gold; ActRII, green; TGF-β3, blue; TGF-βRII, red; BMPRIa, purple. The receptors and their C termini are labeled. Molecular Cell 2003 11, 605-617DOI: (10.1016/S1097-2765(03)00094-7) Copyright © 2003 Cell Press Terms and Conditions

Figure 4 Mutation of Selected ActRII-ECD Residues Inhibits the Ability of Kinase-Deleted ActRII to Function as a Dominant-Negative HepG2 cells were transfected with ActRIItrunc constructs: (1) wild-type, (2) K56A, (3) L61S, (4) V81A, (5) V81Y, (6) F83A, (7) F42A, (8) W60A, or (9) empty vector. Cells were subsequently, treated with vehicle or (A) 10 nM activin A or (B) 10 nM BMP7 and resulting ligand-dependent fold induction of luciferase activity is shown. Molecular Cell 2003 11, 605-617DOI: (10.1016/S1097-2765(03)00094-7) Copyright © 2003 Cell Press Terms and Conditions

Figure 5 Activin and BMP7 Crosslinking to Cell Surface Receptors 293T cells transfected with the indicated receptor constructs were treated with 125I-labeled activin or BMP7, washed, and then crosslinked with DSS. The cells were solubilized, split into two pools, and precipitated with either a type I or type II receptor-specific antibody. (A) Crosslinking to 125I-activin and IP with anti-myc (ActRII). (B) Crosslinking with 125I-activin and IP with anti-FLAG (ALK4). (C) Crosslinking to 125I-BMP7 and IP with anti-FLAG (ActRII). (D) Crosslinking to 125I-BMP7 and IP with anti-myc (ALK2). The full-length ALK2 has no epitope tag so lanes 4, 6, and 8 were precipitated with ALK2-specific antibodies. Molecular Cell 2003 11, 605-617DOI: (10.1016/S1097-2765(03)00094-7) Copyright © 2003 Cell Press Terms and Conditions