pubmed.ncbi.nlm.nih.gov

Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub - PubMed

  • ️Tue Jan 01 2013

Structure of the repulsive guidance molecule (RGM)-neogenin signaling hub

Christian H Bell et al. Science. 2013.

Abstract

Repulsive guidance molecule family members (RGMs) control fundamental and diverse cellular processes, including motility and adhesion, immune cell regulation, and systemic iron metabolism. However, it is not known how RGMs initiate signaling through their common cell-surface receptor, neogenin (NEO1). Here, we present crystal structures of the NEO1 RGM-binding region and its complex with human RGMB (also called dragon). The RGMB structure reveals a previously unknown protein fold and a functionally important autocatalytic cleavage mechanism and provides a framework to explain numerous disease-linked mutations in RGMs. In the complex, two RGMB ectodomains conformationally stabilize the juxtamembrane regions of two NEO1 receptors in a pH-dependent manner. We demonstrate that all RGM-NEO1 complexes share this architecture, which therefore represents the core of multiple signaling pathways.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. Structure of the RGMB-NEO1 complex

(A) Schematic of NEO1 and RGMs. SP indicates signal peptide; IG, Ig-like C2-type 1; TM, transmembrane; CD, C-terminal domain; GPI, glycosylphosphatidylinositol anchor; and vWFD, von Willebrand factor D domain-like. (B) eRGMB ribbon diagram in rainbow coloring (blue, N terminus, red, C terminus). Disulfides (black sticks) are depicted with roman numerals. The autocatalytic cleavage site is marked with asterisks. (C) Schematics of the 2:2 RGMB-NEO1 complex. RGMB is blue and violet; NEO1 is red (FN5), orange (FN6), and green. Interface-buried surface areas (Å2) are shown. (D) Ribbon (left) and surface representation of the 2:2 eRGMB-NEO1FN56 complex. Site-1 and site-2 interfaces are highlighted with boxes. Color coding is as in (C). Right image is 90° rotated around the y axis compared with the left representation.

Fig. 2
Fig. 2. Detailed interactions of the RGMB-NEO1 complex

Color coding is as Fig. 1D. (A) Ribbon representation of the RGMB-NEO1 site-1 complex. The L3 loop of NEO1 is marked. (B and C) Open-book view showing the solvent-accessible surface of the site-1 interface (formed by 17 hydrogen bonds and 147 nonbonded contacts). (B) Interface residues (I, Ile; L, Leu; Q, Gln; T, Thr). Cyan, hydrophilic interactions; yellow, nonbonded contacts. Residues tested by site-directed mutagenesis and functional experiments are labeled. (C) Residue conservation (from nonconserved, white, to conserved, black) based on sequence alignments from vertebrate NEO1 and RGM family members. (D) Ribbon representation of the RGMB-NEO1 site-2 complex. The site-2 interaction uses the RGMB β5-β6 and β10-β11 loop regions contacting the NEO1 FN5 and FN6 domains. K, Lys; V, Val.

Fig. 3
Fig. 3. Biophysical characterization of the RGMB-NEO1 complex

(A to C) SPR equilibrium binding. Different concentrations of eNEO1 (A), NEO1FN56 (B), and eNEO1-L1046E (where E is Glu) (C) were injected over surfaces coupled with eRGMB. RU, response units; Kd, dissociation constant; Bmax, maximum binding capacity; and N/A, not applicable. (D to F) Sedimentation velocity AUC experiments of eRGMB-WT [(D) violet], eRGMB-P206N [(D) blue], NEO1FN56M [(D) red], eRGMB-NEO1 (E), and eRGMB-P206N-NEO1 (F) complexes. Data fitted by using a continuous c(s) function (where s is the sedimentation coefficient) distribution model (solid line). Gaussian peaks contributing to the overall distributions (dotted lines) for eRGMB-NEO1 [(E), root mean square deviation (RMSD) = 0.0038] and eRGMB-P206N-NEO1 [(F), RMSD = 0.0063] complexes. Individual components run as monomeric species. The eRGMB-NEO1FN56M complex shows two major species, indicating both 1:1 and 2:2 complexes. The eRGMB-P206N mutation introduces an N-linked glycan at the site-2 interface. The resulting eRGMB-P206N-NEO1 complex shows a single species corresponding to the 1:1 complex.

Fig. 4
Fig. 4. Functional analysis of site-1 and -2 mutations on RGMB neurite growth inhibitory effects

(A) Representative examples of P9 mouse CGN explants on coverslips coated with RGMB-WT (top left), site-2 mutant RGMB-P206N (top right), site-1 mutant RGMB-A186R (bottom left), or control (bottom right) proteins. Green, βIII-tubulin; red, F-actin; blue, nuclei. (B) Quantification of CGN neurite outgrowth. Distribution of neurite length (short, medium, and long) relative to the control is displayed (total number of explants analyzed for WT, n = 27; P206N, n = 24; A186R, n = 26; control n = 23); error bars are SEM, and *P < 0.01. (C) Model of trans RGMB-NEO1 signaling. RGM ectodomains can be shed by proteolytic or phospholipase activity (open triangle). RGM-binding to preclustered NEO1 results in formation of NEO1 dimers with a defined, signaling-compatible orientation that may be part of a supramolecular clustered state. This arrangement leads to activation of downstream signaling via RhoA (9) (gray lightning bolt). NET1 can inhibit RGM signaling by either simultaneous NEO1 binding or competing with the RGM-NEO1 interaction. The gray box highlights the RGM-NEO1 signaling hub observed in the crystal structure.

Similar articles

Cited by

References

    1. Monnier PP, et al. Nature. 2002;419:392–395. - PubMed
    1. Niederkofler V, Salie R, Sigrist M, Arber S. J. Neurosci. 2004;24:808–818. - PMC - PubMed
    1. Mirakaj V, et al. Proc. Natl. Acad. Sci. U.S.A. 2011;108:6555–6560. - PMC - PubMed
    1. Xia Y, et al. J. Immunol. 2011;186:1369–1376. - PMC - PubMed
    1. Papanikolaou G, et al. Nat. Genet. 2004;36:77–82. - PubMed

Publication types

MeSH terms

Substances