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. 2015 Mar 24;112(12):3710-5.
doi: 10.1073/pnas.1501303112. Epub 2015 Mar 6.

Structure of bone morphogenetic protein 9 procomplex

Li-Zhi Mi  1 , Christopher T Brown  2 , Yijie Gao  2 , Yuan Tian  3 , Viet Q Le  3 , Thomas Walz  4 , Timothy A Springer  5
Affiliations

Structure of bone morphogenetic protein 9 procomplex

Li-Zhi Mi et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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) BMP9 and TGF-β1 prodomains shown in cartoon after superimposition. 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 pro-BMP9 (K). Structures are superimposed on the GF monomer. Colors are as in A, B, E, and F. Key residues are shown in stick.
Fig. 2.
Fig. 2.
Sequence alignments. (A) Sequences aligned structurally with SSM (26). Structurally aligned residues are in uppercase; lowercase residues are not structurally aligned. Residues with missing density are in italics. Decadal residues are marked with black dots. Residues with more than 50% solvent accessible surface area buried in prodomain–GF interfaces are marked with gold dots. Secondary structural elements or named regions are shown with lines. Long black arrow marks cleavage sites between the prodomain and GF; short black arrows mark putative PC cleavage sites after the α1-helix and in the α5-helix. Stars mark HHT-like mutation sites. (B) Sequence alignment of representative TGF-β family members in the prodomain α1-helix (overlined for TGF-β1).
Fig. 3.
Fig. 3.
The prodomain α2-helix. (A and B) Superimpositions based on the arm domain (A) or on the GF (B), which are shown as Cα traces with the α2-helix in cartoon. (C and D) Prodomain α2-helix environments in pro-BMP9 (C) and pro-TGF-β1 (D) shown after superimposition on the prodomain α2-helix and GF β6 and β7-strands. Key interacting residues are shown in stick.
Fig. 4.
Fig. 4.
Binding of BMP9 to the prodomain and receptors. (A and B) Representative ITC data at 15 °C adding material either to the BMP9 dimer (A) or prodomain (B) in the calorimetry cell. (Upper) Baseline-corrected raw data. (Lower) Integrated heats fit to the independent-binding site model. (C and D) Differentiation of C2C12 cells measured by alkaline phosphatase production. (C) Comparison of BMP9 and pro-BMP9. (D) Inhibition of 1 nM BMP9 by GCN4-linked, oligomeric BMP9 prodomain and native prodomain. (E and F) EC50 values of BMP9 and pro-BMP9 for Fc-fused type I (E) or type II (F) receptor ectodomains measured using quantitative ELISA. Data are plotted as the fraction of maximal bound in each experiment and fit to the equation of fractional saturation. Graphs show average of triplicates in one experiment; numerical values show mean EC50 and sd from three such experiments. (G and H) Superimpositions on pro-BMP9 of the BMP9–receptor complex (17) (G) and BMP2-crossveinless2 complex (H) in identical orientations. For clarity, BMP in receptor and inhibitor complexes is omitted, and the α5-helix is transparent in H.
Fig. 5.
Fig. 5.
Models for pro-BMP9 structures and binding to receptors. Models for the open-armed, nonlatent pro-BMP9 conformation characterized here (1), the proposed (dashed lines) pro-TGF-β1–like cross-armed conformation of pro-BMP9 (2), and stepwise binding (18) to type I (3) and type II receptors (4).
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