Structural basis of semaphorin-plexin signalling

Abstract

Cell-cell signalling of semaphorin ligands through interaction with plexin receptors is important for the homeostasis and morphogenesis of many tissues and is widely studied for its role in neural connectivity, cancer, cell migration and immune responses. SEMA4D and Sema6A exemplify two diverse vertebrate, membrane-spanning semaphorin classes (4 and 6) that are capable of direct signalling through members of the two largest plexin classes, B and A, respectively. In the absence of any structural information on the plexin ectodomain or its interaction with semaphorins the extracellular specificity and mechanism controlling plexin signalling has remained unresolved. Here we present crystal structures of cognate complexes of the semaphorin-binding regions of plexins B1 and A2 with semaphorin ectodomains (human PLXNB1(1-2)-SEMA4D(ecto) and murine PlxnA2(1-4)-Sema6A(ecto)), plus unliganded structures of PlxnA2(1-4) and Sema6A(ecto). These structures, together with biophysical and cellular assays of wild-type and mutant proteins, reveal that semaphorin dimers independently bind two plexin molecules and that signalling is critically dependent on the avidity of the resulting bivalent 2:2 complex (monomeric semaphorin binds plexin but fails to trigger signalling). In combination, our data favour a cell-cell signalling mechanism involving semaphorin-stabilized plexin dimerization, possibly followed by clustering, which is consistent with previous functional data. Furthermore, the shared generic architecture of the complexes, formed through conserved contacts of the amino-terminal seven-bladed β-propeller (sema) domains of both semaphorin and plexin, suggests that a common mode of interaction triggers all semaphorin-plexin based signalling, while distinct insertions within or between blades of the sema domains determine binding specificity.

Figures

Figure 1. The semaphorin–plexin complexes share a common architecture

a, Schematic domain organization of human PLXNB1, mouse PlxnA2, human SEMA4D and mouse Sema6A. PLXNB1 contains an additional mucin-like domain inserted into the PSI2 domain. SP, signal peptide; TM, transmembrane. The domains included in the crystallization constructs are coloured. b, Ribbon representation of PlxnA21–4 ‘rainbow’ colour ramped from blue (N terminus) to red (C terminus) with the β-propeller blades numbered. N-linked glycans are shown in magenta ball-and-stick representation and the 14 disulphide bridges (black stick presentation) are marked with Roman numbering. c, Multi-angle light scattering indicates an experimental molecular mass (black line) of 83.7 ± 0.8 kDa for PlxnA21–4 (green line; elution profile, axis not shown) as observed by SDS–PAGE (inset) and in agreement with the theoretical molecular mass for a monomer (85 kDa). d, Ribbon representation (left panel), cartoon drawing (middle panel) and surface representations with individual protein chains indicated by an outline (right panel) of the PLXNB11–2–SEMA4Decto and PlxnA21–4–Sema6Aecto complexes. Domains are coloured as in Fig. 1a.

Figure 2. Similar characteristics mediate the semaphorin–plexin interactions

a, Ribbon representation of the pseudo twofold arrangement of the interacting semaphorin and plexin sema domains. b, An opened view showing the semaphorin–plexin interface (green), the semaphorin homodimer interface (yellow) and interface mutants used in biophysical and cellular assays (red) (top panel). Semaphorin and plexin are colour-coded according to residue conservation (from non-conserved, white, to conserved, black) based on alignments containing sequences from all vertebrate semaphorin and plexin classes (middle panel). Semaphorin and plexin coloured by electrostatic potential from red (−8 kbT/ec) to blue (8 kbT/ec) (bottom panel). In both complexes the interface consists of conserved complementary charged patches.

Figure 3. Bivalent interaction is critical for semaphorin–plexin-induced cell-cell signalling

a, SPR equilibrium experiments of PLXNB11–2–SEMA4Decto (top panels; wild-type SEMA4Decto and monomerized SEMA4Decto(F244N/F246S)) and PlxnA21–4–Sema6Aecto (bottom panels; Sema6Aecto over PlxnA21–4 and reversed orientation, see also Supplementary Fig. 7). RU, response units. b, MALS analyses indicate molecular masses (black lines) of 174 ± 2 kDa and 92 ± 1 kDa for SEMA4Decto and SEMA4Decto(F244N/F246S), respectively (elution profiles; green and blue lines, axis not shown). c, d, Cos-7 cell collapse assay showing representative images of non-collapsed cells (c, left panel) and SEMA4Decto-induced collapsed cells (c, right panel). Scale bar, 40 μm. e, EGL explants (green) grown on NIH3T3 cells (red) without (left panel) or with (right panel) Sema6A expression show the migration of post-mitotic granular neurons. WT, wild type. Scale bar, 200 μm. f, Quantification of migrating post-mitotic neurons from cultured EGL explants of either PlxnA2+/− or PlxnA2−/− mice grown on wild type (shaded) or Sema6A expressing cells (hatched). (**P ≤ 0.005 by unpaired t-test). g, Model for semaphorin-stabilized plexin signalling. Binding of semaphorin stabilizes plexin dimerization, sufficient plexin ectodomain flexibility may enable plexin-to-plexin cis interaction in their membrane-proximal regions (upper panel) and seed further oligomerization. Possibly, dimerization is preceded by a ‘switch-blade’ conformational change in the plexin ectodomain (lower panel) exposing cis interaction sites leading to extracellular clustering. Two types of initial binding events (dotted enclosures) could result in the dimer and cluster architecture of either the upper or lower panel. The precise arrangement of the cytoplasmic region in the active state triggered by extracellular clustering cannot be specified.