Structure of bone morphogenetic protein 9 procomplex Li-Zhi Mia,b,c, Christopher T. Brownd, Yijie Gaod, Yuan Tiana,b, Viet Q. Lea,b, Thomas Walze,f, and Timothy A. Springera,b,1 aDepartment of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115; bProgram in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, MA 02115; cDepartment of Molecular and Structural Biology, School of Life Sciences, Tianjin University, Tianjin 300072, China; dGlobal BioTherapeutics Technologies, Pfizer Worldwide Research and Development, Cambridge, MA 02139; and eHoward Hughes Medical Institute and fDepartment of Cell Biology, Harvard Medical School, Boston, MA 02115 Contributed by Timothy A. Springer, January 27, 2015 (sent for review January 2, 2015; reviewed by Daniel Rifkin and Lynn Y. Sakai) Bone morphogenetic proteins (BMPs) belong to the TGF-β family, the structure of pro-BMP9 and compare it to the previously de- whose 33 members regulate multiple aspects of morphogenesis. scribed, cross-armed conformation of pro-TGF-β1 (10). Although TGF-β family members are secreted as procomplexes containing a a member of the BMP subfamily and possessing chondrogenic small growth factor dimer associated with two larger prodomains. and osteogenic activity, BMP9 is expressed in liver and is required As isolated procomplexes, some members are latent, whereas most for properly organized blood and lymphatic vascular development are active; what determines these differences is unknown. Here, (11, 12). Mutations in the prodomain of BMP9, in its receptor studies on pro-BMP structures and binding to receptors lead to β Alk1, and in its coreceptor endoglin cause phenotypically over- insights into mechanisms that regulate latency in the TGF- family lapping hereditary hemorrhagic telangiectasias (13–15). and into the functions of their highly divergent prodomains. The Here, we reveal surprising open-armed conformations of pro- observed open-armed, nonlatent conformation of pro-BMP9 and BMPs 7 and 9. We propose that binding to interactors in the pro-BMP7 contrasts with the cross-armed, latent conformation of β matrix may regulate transition between open-armed and cross- pro-TGF- 1. Despite markedly different arm orientations in pro- β BMP and pro-TGF-β, the arm domain of the prodomain can simi- armed conformations in the TGF- family and that these con- larly associate with the growth factor, whereas prodomain ele- formations regulate GF latency. ments N- and C-terminal to the arm associate differently with Results the growth factor and may compete with one another to regulate Structures of BMP Procomplexes. latency and stepwise displacement by type I and II receptors. Se- In marked contrast to the cross- quence conservation suggests that pro-BMP9 can adopt both cross- armed, ring-like conformation of pro-TGF-β1 (10), crystal armed and open-armed conformations. We propose that interactors structures of natively glycosylated pro-BMP9 reveal an un- in the matrix stabilize a cross-armed pro-BMP conformation and expected, open-armed conformation (Fig. 1 A and B and Table regulate transition between cross-armed, latent and open-armed, S1). All negative stain EM class averages show an open-armed nonlatent pro-BMP conformations. conformation for pro-BMP9 (Fig. 1C and Fig. S1) and a similar, although less homogenous, open-armed conformation for pro- embers of the TGF-β family including bone morphoge- BMP7 (Fig. 1D and Fig. S2). Crystal structure experimental Mnetic proteins (BMPs) are biosynthesized and processed electron density is excellent (Fig. S3) and allows us to trace the into complexes between large prodomains and smaller, C-ter- complete structure of each pro-BMP9 arm domain (residues 63– minal mature growth factor (GF) domains that are separated by 258; Fig. 1E). As in pro-TGF-β1, the arm domain has two β-sheets proprotein convertase (PC) (furin) cleavage sites. In the original that only partially overlap. Hydrophobic, nonoverlapping portions isolation of proteins responsible for BMP activity, bone was first of the β-sheets are covered by meandering loops and the α4-helix demineralized with 0.5 M HCl. The resulting residual matrix was (Fig. 1 E and F). Comparison of pro-BMP9 and pro-TGF-β1arm – extracted with 6 M urea or 4 M guanidine HCl (1 3). During domains defines a conserved core containing two four-stranded subsequent purification under largely denaturing conditions, the β-sheets and the α4-helix (labeled in black in Fig. 1 E and F). GF domains were separated from their prodomains. Therefore, One of the BMP9 arm domain β-sheets joins a finger-like little attention was paid to the potential existence of BMP pro- β-sheet in the GF to form a super β-sheet (Fig. 1 A and G). Each complexes. However, evidence exists that BMP prodomains GF monomer has a hand-like shape. The two BMP9 GF hands contribute to maintaining BMP GF domains inactive or latent in vivo. For example, early studies showed a 60-fold increase in total BMP activity during the first two purification steps fol- Significance lowing extraction of the BMP, which was interpreted as puri- fication of BMP away from an inhibitor (2). This finding is Bone morphogenetic protein (BMP) activity is regulated by consistent with the presence of largely latent complexes between prodomains. Here, structures of BMP procomplexes reveal an BMPs, their prodomains, and extracellular matrix components in open-armed conformation. In contrast, the evolutionarily re- the insoluble residual matrix from which BMPs were purified. In lated, latent TGF-β1 procomplex is cross-armed. We propose agreement with a regulatory role for the prodomain, mutations that in the TGF-β and BMP family, conversion between cross- of secondary PC sites within the prodomain perturb embryonic armed and open-armed conformations may regulate release development in insects and vertebrates, suggesting that prodo- and activity of the growth factor. mains of several BMPs remain associated with GFs after secre- tion and regulate the distance over which BMPs signal (4–7). An Author contributions: T.A.S. designed research; L.-Z.M., C.T.B., Y.G., Y.T., V.Q.L., T.W., and T.A.S. performed research; L.-Z.M., C.T.B., Y.G., Y.T., V.Q.L., T.W., and T.A.S. analyzed important role for the prodomain in development is also illustrated data; and L.-Z.M., C.T.B., Y.T., V.Q.L., and T.A.S. wrote the paper. by prodomain mutations, including in secondary PC cleavage sites, Reviewers: D.R., NYU Langone Medical Center; and L.Y.S., Shriners Hospitals for Children. that cause human diseases (5, 7). The authors declare no conflict of interest. Pro-TGF-β is latent; however, when overexpressed as recom- Data deposition: The atomic coordinates and structure factors have been deposited in the binant proteins, most BMPs are active. Although noncovalently Protein Data Bank, www.pdb.org (PDB ID codes 4YCG and 4YCI). associated with their GF after secretion, the prodomains of most 1To whom correspondence should be addressed. Email: [email protected]. BMPs do not bind strongly enough to prevent GF from binding edu. to receptors and signaling (8, 9). To better understand such This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. differences among members of the TGF-β family, we examine 1073/pnas.1501303112/-/DCSupplemental. 3710–3715 | PNAS | March 24, 2015 | vol. 112 | no. 12 www.pnas.org/cgi/doi/10.1073/pnas.1501303112 Downloaded by guest on September 24, 2021 A B Pro- Bowtie Pro- domain domain Prodomain Prodomain RGD RGD Arm Arm Arm domain domain domain Arm Pro- Pro- domain β1 BMP9 α2 TGF-β1 α2 α2 β1 β7 α5 α5 α2 β-finger β7 β6 β6 β-finger α5 Growth Growth Strait- Latency factor factor jacket lasso Latency lasso α1 α1 C D Growth Growth Pro-BMP9 Pro-BMP7 factor Cys factor linkage E248 I K393 α5 F BMP9 0.88 0.88 0.86 TGF-β1 K350 Bowtie Y396 E BMP9 β9 β8 C196 M252 C194 C214 W322 RGD β4 C133 β9’ H255 β4 β3 W319 β5 β2 β5 Arm β7 β2 β7 β1' domain β6 Arm β6 α4 α4 domain β3 J β3 α3 β10 β10 TGF-β1 β1 α2 β1 α2 Fastener L28 α5 Y339 α1 Latency W281 lasso α1 I24 I20 α5 G H I17 BMP9 TGF-β1 W279 R21 Pro- β10 Pro- β10 domain 1 β1 domain 1 K β1 β-finger α2 α5 α2 α5 β7 β7 BMP9 α1 α5 Cross- β6 β6 β-finger armed Y383 L47 model Latency F43 lasso M39 Growth α3 W322 factor 2 α1 Growth Growth Growth factor 2 L36 factor 1 factor 1 W319 K40 Fig. 1. Structures. (A and B) Cartoon diagrams of pro-BMP9 (A) and pro-TGF-β1 (10) (B) with superimposition on GF dimers. Disulfides (yellow) are shown in stick. (C and D) Representative negative-stain EM class averages of pro-BMP9 (C) and pro-BMP7 (D). Best correlating projections of the pro-BMP9 crystal structure with their normalized cross-correlation coefficients are shown below class averages. (Scale bars, 100 Å.) (E and F)BMP9andTGF-β1 prodomains shown in cartoon after su- perimposition. Core arm domain secondary structural elements are labeled in black and others in red. Helices that vary in position between cross- and open-armed conformations are color-coded. Spheres show Cys S atoms. (G–K) Prodomain–GF interactions in pro-BMP9 (G and I), pro-TGF-β1(H and J), and a model of cross-armed BIOPHYSICS AND pro-BMP9 (K). Structures are superimposed on the GF monomer. Colors are as in A, B, E,andF. Key residues are shown in stick. COMPUTATIONAL BIOLOGY Mi et al. PNAS | March 24, 2015 | vol.