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Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein

This article has been updated


Repulsive guidance molecules (RGMs) control crucial processes including cell motility, adhesion, immune-cell regulation and systemic iron metabolism. RGMs signal via the neogenin (NEO1) and the bone morphogenetic protein (BMP) pathways. Here, we report crystal structures of the N-terminal domains of all human RGM family members in complex with the BMP ligand BMP2, revealing a new protein fold and a conserved BMP-binding mode. Our structural and functional data suggest a pH-linked mechanism for RGM-activated BMP signaling and offer a rationale for RGM mutations causing juvenile hemochromatosis. We also determined the crystal structure of the ternary BMP2–RGM–NEO1 complex, which, along with solution scattering and live-cell super-resolution fluorescence microscopy, indicates BMP-induced clustering of the RGM–NEO1 complex. Our results show how RGM acts as the central hub that links BMP and NEO1 and physically connects these fundamental signaling pathways.

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Figure 1: Structure of the BMP2–RGM complex.
Figure 2: Interaction determinants of the BMP2–RGM complex.
Figure 3: The mode of RGM-BMP2 interactions is conserved in RGMA, RGMB and RGMC.
Figure 4: RGMs and BMPR1A share a common binding site on BMP2.
Figure 5: Structure of the ternary BMP2–RGM–NEO1 complex.
Figure 6: BMP2-mediated clustering of RGM–NEO1 complexes.

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Change history

  • 14 May 2015

    In the version of this supplementary file originally posted online on 4 May 2015, the title and legend of Supplementary Figure 7 reproduced part of the title and legend from Supplementary Figure 6, and descriptions of panels c–f were missing. The errors have been corrected in this file as of 14 May 2015.


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We thank the staff of beamlines I03, I04 and I04-1 at the Diamond Light Source (X-ray diffraction data, proposal MX-10627), BM29 at the European Synchrotron Radiation Facility (SAXS data) and the Cellular Imaging Core at the Wellcome Trust Centre for Human Genetics (TIRF and dSTORM data) for assistance; T. Walter and K. Harlos for help with crystallization; R. Robinson and G. Sutton for help with MALS; and A.R. Aricescu and D.I. Stuart for reading the manuscript. This work was supported by Cancer Research UK (C20724/A14414 (C.S.)) and the Wellcome Trust (097301/Z/11/Z (E.G.H.)). Further support from the Wellcome Trust core award grant 090532/Z/09/Z (C.S.) and the Wellcome Trust multi-user equipment grant 101584MA (S.P.-P. and C.S.) are acknowledged. E.G.H. is funded by a Wellcome Trust PhD Studentship. J.E. is supported as a Marie-Curie Postdoctoral Fellow (FP7-328531). S.P.-P. is supported as a Nuffield Department of Medicine Leadership Fellow. C.S. is supported as a Cancer Research UK Senior Research Fellow.

Author information




C.S. designed and supervised the project. E.G.H. and C.H.B. cloned all RGM, NEO1 and BMP constructs. E.G.H., B.B. and C.H.B. performed protein expression and purification, and E.G.H. crystallized the proteins. E.G.H. and C.S. collected the data and solved and refined the crystal structures. E.G.H. and B.B. carried out SPR and luciferase experiments, and E.G.H. performed the MALS experiments. SAXS data were collected by J.E. and E.G.H., and J.E. conducted all subsequent SAXS data processing. E.G.H., B.B. and S.P.-P. collected the imaging data, and S.P.-P. completed the dSTORM data processing. C.S. and E.G.H. wrote the paper, and all authors discussed the results and commented on the paper.

Corresponding author

Correspondence to Christian Siebold.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Sequence alignments of BMP and RGM family members.

(a) Sequence alignment of human BMP family members. Numbering corresponds to the full length human BMP2; residues of BMP2 molecule 1 forming hydrophilic interactions to RGMC are highlighted in dark blue and non-bonded contacts in light blue. Residues of BMP2 molecule 2 forming non-bonded contacts are shown in red. Disulfide bridges are indicated by Roman Numerals. (b) Sequence alignment of the N-terminal domain of RGM family members. Numbering is that of the full length human RGMC. Secondary structure assignments for RGMAND (blue), RGMBND (yellow) and RGMCND (orange) are displayed above the alignment. RGM residues forming hydrophilic interactions with BMP2 are highlighted in dark blue, exclusively formed by RGMA in salmon and exclusively formed by RGMC in green. Non-bonded contact residues interacting with BMP2 are depicted in magenta. Disulfide bridges are indicated by Roman Numerals. Asterisks (*) indicate disease-related residues identified in human RGMC. hRGM: human RGM, mRGM: mouse RGM, zRGM: zebrafish RGM, xRGM: Xenopus laevis RGM.

Supplementary Figure 2 Electron density maps of the RGMC–BMP2 complex.

(a) Initial electron density map, at 2.35 Å resolution, of the RGMCND–BMP2 complex after molecular replacement (using only the BMP2 dimer as search model) in PHASER (McCoy, A. J. et al. (2007) J Appl Crystallogr 40, 658-674) and density modification in PARROT (Cowtan, K. (2010) Acta Crystallogr D 66, 470-478). Contour level is 1.0 σ. The final refined RGMCND–BMP2 model is represented as ribbon (RGMC: blue and green; BMP2: yellow and magenta). (b-c) Close-up view onto the RGMC–BMP2 interface. Protein chains are depicted in stick representation. Color coding is as in (a). (b) Initial electron density map as described in (a). (c) SigmaA-weighted 2FO-FC map after the final round of refinement from autoBUSTER (BUSTER, version 2.8.0., Cambridge, 2011) contoured at 1.0 σ.

Supplementary Figure 3 RGMCND structure and comparison.

(a) Topology diagram of RGMCND (adapted from PDBSUM ( in rainbow coloring (N-terminus: blue; C-terminus: red). N- and C-termini and residue numbers are shown. (b) Cartoon representation of the RGMCND structure. Color coding is as in (a). RGMND forms a compact three helix bundle fold, stabilized by three disulfide bonds, supported by a hydrophobic core including residues from all three helices, depicted as sticks. Right panel rotated 90° around the x-axis compared to left panel. Disulfide bridges are numbered in Roman numerals. The disordered α1–α2 loop is shown as a dotted line. (c-g) Structural comparison of RGMCND using the PDBefold server ( RGMCND ((c), cyan) shares the closest structural similarity to the human MTCP1 oncogene ((d), pdb 2HP8 (Barthe, P. et al. (1997) J Mol Biol 274, 801-815), slate; r.m.s.d. of 2.78 Å for 60 equivalent Cα positions, sequence identity: 5%), the MIT domain of VPS4-like ATPases ((e), pdb 2V6Y (Obita, T. et al. (2007) Nature 449, 735-739), magenta, r.m.s.d. of 2.47 Å for 60 equivalent Cα positions, sequence identity: 3%) and the complement inhibitor EHP from S. aureus ((f), pdb 2NOJ (Hammel, M. et al. (2007). J Biol Chem 282, 30051-30061), yellow, r.m.s.d. of 2.34 Å for 54 equivalent Cα positions, sequence identity: 6%). The position of disulfide bridge II in RGMND is conserved in MTCP1 and highlighted by an asterisk. A structural superposition is shown in (g). The two views are related by a rotation of 90° around the x-axis.

Supplementary Figure 4 SPR equilibrium binding experiments.

(a-l) Binding of different constructs and mutants of RGMA, RGMB and RGMC, respectively, to BMP2. (m-r) Binding of BMP receptor ectodomain constructs to BMP2 ligand (m-o) or to eRGMB (p-r). For (k) the eRGMB-NEO1FN56 complex was purified via size exclusion chromatography prior to SPR. Graphs show a plot of the equilibrium binding response (response units (RU)) against concentration of the used analytes. All experiments were performed in duplicate. Binding constants (Kd) are given as mean with the error representing the standard error of the mean (n=2 technical repeats) Fits are shown as lines. Sensorgrams are shown..Binding constants (Kd), Bmax and surface response units (Bsurface) are indicated below the graphs.

Supplementary Figure 5 Analysis of the effects of eBMPR1A and eRGMB on SMAD-mediated transcriptional activation by BMP-responsive reporter (BRE-LUC).

(a-c) LLC-PK1 cells (a, b) or C2C12 cells (c) were stimulated with either buffer control or different concentrations of BMP2: 25 nM (a), 6 nM BMP2 (b); or 10 nM BMP2 (c). BMP2 was pre-incubated with 0.4, 1.6, 6.3, 25 or 100 X molar excess of eBMPR1A or eRGMB. Average BRE-Luc relative response was calculated for each condition from two independent experiments. For (a): Control n = 4, +25 nM BMP2 n = 12, all others n= 8. For (b): Control and + 6 nM BMP2 n = 33, all eBMPR1A n = 41, all eRGMB n = 32. For (c): Control n= 6, + 10 nM BMP2 n = 18, all others n = 12. Where n = cell cultures. Error bars are s.e.m., dotted line indicates average increase in relative luciferase response induced by the respective BMP2 concentration in each case.

Supplementary Figure 6 Interface analysis and comparison of the ternary RGMB–NEO1–BMP2 complex to the binary RGMB–NEO1 and RGMB–BMP2 complexes.

(a) Cartoon representations of the ternary RGMB–NEO1–BMP2 complex (grey) and superimposed binary RGMB–NEO1 (orange and red) and RGMB–BMP2 (orange and blue) complexes. Superposition of the binary RGMBND–BMP2 complex from this study yields in an r.m.s.d. of 0.799 Å for 328 equivalent Cα positions. Using the previously determined structure of the binary NEO1FN56–eRGMB complex (pdb 4BQ6, site-1 interface (Bell, C. H. et al. (2013) Science 341(6141), 77-80) an r.m.s.d. of 0.511 Å for 368 equivalent Cα positions was achieved when compared with the ternary RGMB–NEO1–BMP2 complex. (b) Table showing the analysis of the interfaces highlighted in (a). Whereas the binary BMP2–BMP2, BMP2–RGMBND and NEO1FN56–RGMBCD interfaces show values expected for a physiological interaction, the BMP2-NEO1FN5 interface has a at least 3-times smaller buried interface area and significantly lower score. ashape complementary calculated with program sc (Lawrence M. C. et al. (1993) J Mol Biol 234, 946-950); btotal buried surface area (from PISA (Krissinel E. et al. (2007) J Mol Biol 372, 774-797 (1)); csolvation free energy gain upon formation of the interface1; dnumber of interfacing residues in the complex1; enumber of potential hydrogen bonds across the interface1; fcomplexation Significance Score indicating how significant the interface is for assembly formation1; gbrackets indicate the percentage of buried interface area compared to the overall surface of the free molecules; hthe BMP2-BMP2 interface also includes a intermolecular disulfide bridge that is not included in the number of interacting residues.

Supplementary Figure 7 SAXS solution structures of NEO1FN56M, eRGMB, eRGMB–NEO1FN56M and BMP2–eRGMB–NEO1FN56M.

(a-b) SEC of the BMP2-eRGMB-NEO1FN56M complex formed by saturating BMP2 and eRGMB with a molar excess of NEO1FN56M. The SEC elution profile (a) and the corresponding SDS-PAGE of the fractions under reducing conditions (b) are shown. The first peak (I) corresponds to the ternary BMP2-eRGMB-NEO1FN56M complex, the second (II) to the binary eRGMB-NEO1FN56M complex, and the third (III) to uncomplexed NEO1FN56M, which was added in a 1.5 molar excess. MW: Sigma Molecular Weight Marker S8445. The asterisks mark the two RGM products resulting from autocatalytic cleavage7. (c-f) SAXS analysis of NEO1FN56M (c), eRGMB (d), eRGMB-NEO1FN56M (e) and BMP2-eRGMB-NEO1FN56M (f) after isolation by SEC. Experimental scattering curves (black) and calculated scattering patterns (red) are shown to a maximal momentum transfer of q = 0.35 Å−1. The fitting residuals of the experimental scattering curves and calculated scattering patterns are displayed in the lower left insets. The upper right insets show the experimental (black) and calculated (red) normalized pair distance distribution (P(r)) function. The lower right insets display the experimental (black) and calculated (red) Guinier region. The shaded area indicates the range of fitting for RG analysis (RG•S ≤ 1.3). The fit of the calculated scattering pattern (χ2), the derived maximum intra-particle diameter (DMAX), the radius of gyration (RG), and the molecular weight derived from the volume of correlation metric VC (MWVc) are annotated.

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Healey, E., Bishop, B., Elegheert, J. et al. Repulsive guidance molecule is a structural bridge between neogenin and bone morphogenetic protein. Nat Struct Mol Biol 22, 458–465 (2015).

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