Structure of bone morphogenetic protein 9 procomplex

Li Mi

Christopher Brown

Yijie Gao

Yuan Tian

Viet Le

Thomas Walz

Timothy Springer

Supplementary Material

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^{Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115;}

^{Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, MA, 02115;}

^{Department of Molecular and Structural Biology, School of Life Sciences, Tianjin University, Tianjin 300072, China;}

^{Global BioTherapeutics Technologies, Pfizer Worldwide Research and Development, Cambridge, MA, 02139; and}

^{Howard Hughes Medical Institute and}

^{Department of Cell Biology, Harvard Medical School, Boston, MA, 02115}

^{To whom correspondence should be addressed. Email:}ude.dravrah.snerdlihc@regnirps.yhtomit.

Contributed by Timothy A. Springer, January 27, 2015 (sent for review January 2, 2015; reviewed by Daniel Rifkin and Lynn Y. Sakai)

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 data; and L.-Z.M., C.T.B., Y.T., V.Q.L., and T.A.S. wrote the paper.

Reviewers: D.R., NYU Langone Medical Center; and L.Y.S., Shriners Hospitals for Children.

Contributed by Timothy A. Springer, January 27, 2015 (sent for review January 2, 2015; reviewed by Daniel Rifkin and Lynn Y. Sakai)

Significance

Bone morphogenetic protein (BMP) activity is regulated by prodomains. Here, structures of BMP procomplexes reveal an open-armed conformation. In contrast, the evolutionarily related, latent TGF-β1 procomplex is cross-armed. We propose that in the TGF-β and BMP family, conversion between cross-armed and open-armed conformations may regulate release and activity of the growth factor.

Significance

Abstract

Bone morphogenetic proteins (BMPs) belong to the TGF-β family, whose 33 members regulate multiple aspects of morphogenesis. TGF-β family members are secreted as procomplexes containing a small growth factor dimer associated with two larger prodomains. As isolated procomplexes, some members are latent, whereas most are active; what determines these differences is unknown. Here, studies on pro-BMP structures and binding to receptors lead to insights into mechanisms that regulate latency in the TGF-β family and into the functions of their highly divergent prodomains. The observed open-armed, nonlatent conformation of pro-BMP9 and pro-BMP7 contrasts with the cross-armed, latent conformation of pro-TGF-β1. Despite markedly different arm orientations in pro-BMP and pro-TGF-β, the arm domain of the prodomain can similarly associate with the growth factor, whereas prodomain elements N- and C-terminal to the arm associate differently with the growth factor and may compete with one another to regulate latency and stepwise displacement by type I and II receptors. Sequence conservation suggests that pro-BMP9 can adopt both cross-armed and open-armed conformations. We propose that interactors in the matrix stabilize a cross-armed pro-BMP conformation and regulate transition between cross-armed, latent and open-armed, nonlatent pro-BMP conformations.

Abstract

Members of the TGF-β family including bone morphogenetic proteins (BMPs) are biosynthesized and processed into complexes between large prodomains and smaller, C-terminal mature growth factor (GF) domains that are separated by proprotein convertase (PC) (furin) cleavage sites. In the original isolation of proteins responsible for BMP activity, bone was first demineralized with 0.5 M HCl. The resulting residual matrix was extracted with 6 M urea or 4 M guanidine HCl (1 –3). During subsequent purification under largely denaturing conditions, the GF domains were separated from their prodomains. Therefore, little attention was paid to the potential existence of BMP procomplexes. However, evidence exists that BMP prodomains 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 following extraction of the BMP, which was interpreted as purification of BMP away from an inhibitor (2). This finding is consistent with the presence of largely latent complexes between BMPs, their prodomains, and extracellular matrix components in the insoluble residual matrix from which BMPs were purified. In agreement with a regulatory role for the prodomain, mutations of secondary PC sites within the prodomain perturb embryonic development in insects and vertebrates, suggesting that prodomains of several BMPs remain associated with GFs after secretion and regulate the distance over which BMPs signal (4 –7). An important role for the prodomain in development is also illustrated by prodomain mutations, including in secondary PC cleavage sites, that cause human diseases (5, 7).

Pro-TGF-β is latent; however, when overexpressed as recombinant proteins, most BMPs are active. Although noncovalently associated with their GF after secretion, the prodomains of most BMPs do not bind strongly enough to prevent GF from binding to receptors and signaling (8, 9). To better understand such differences among members of the TGF-β family, we examine the structure of pro-BMP9 and compare it to the previously described, cross-armed conformation of pro-TGF-β1 (10). Although a member of the BMP subfamily and possessing chondrogenic and osteogenic activity, BMP9 is expressed in liver and is required for properly organized blood and lymphatic vascular development (11, 12). Mutations in the prodomain of BMP9, in its receptor Alk1, and in its coreceptor endoglin cause phenotypically overlapping hereditary hemorrhagic telangiectasias (13 –15).

Here, we reveal surprising open-armed conformations of pro-BMPs 7 and 9. We propose that binding to interactors in the matrix may regulate transition between open-armed and cross-armed conformations in the TGF-β family and that these conformations regulate GF latency.

Footnotes

The authors declare no conflict of interest.

Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 4YCG and 4YCI).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1501303112/-/DCSupplemental.

Footnotes

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