Neuropilins lock secreted semaphorins onto plexins in a ternary signaling complex

Abstract

Co-receptors add complexity to cell-cell signaling systems. The secreted semaphorin 3s (Sema3s) require a co-receptor, neuropilin (Nrp), to signal through plexin As (PlxnAs) in functions ranging from axon guidance to bone homeostasis, but the role of the co-receptor is obscure. Here we present the low-resolution crystal structure of a mouse semaphorin-plexin-Nrp complex alongside unliganded component structures. Dimeric semaphorin, two copies of plexin and two copies of Nrp are arranged as a dimer of heterotrimers. In each heterotrimer subcomplex, semaphorin contacts plexin, similar to in co-receptor-independent signaling complexes. The Nrp1s cross brace the assembly, bridging between sema domains of the Sema3A and PlxnA2 subunits from the two heterotrimers. Biophysical and cellular analyses confirm that this Nrp binding mode stabilizes a canonical, but weakened, Sema3-PlxnA interaction, adding co-receptor control over the mechanism by which receptor dimerization and/or oligomerization triggers signaling.

Figure 1. Nrp1 locks Sema3A and PlxnA2 together

a, Schematic domain organisation of mouse Sema3A, PlxnA2 and Nrp1. SS: signal sequence, TM: transmembrane region, l: linker, t: tail, GAP: GTPase activating protein, RBD: Rho GTPase-binding domain. Arrows indicate furin processing sites in Sema3A. The domains included in the crystallization constructs are colored. b, Ribbon representation of Nrp1_1-4, Ca²⁺ ions shown as orange spheres and N-linked glycans shown in grey stick representation (top panel). Detailed view of the two Ca²⁺ binding sites in Nrp1 with coordinating residues in stick representation and waters as small red spheres (bottom panels). c, SPR equilibrium experiments indicate no or very little interaction between Sema3A_S-P-PlxnA2_1-4, direct interaction between Sema3A_S-P-Nrp1_1-4 and PlxnA2_1-4-Nrp1_1-4 and an added effect upon interaction of all three (Sema3A_S-P-PlxnA2_1-4-Nrp1_1-4) components.

Figure 2. Complex architecture and interfaces are conserved and validated

a, The Sema3A_S-F-PlxnA2_1-4-Nrp1_1-4 complex with Sema3A and PlxnA2 in ribbon representation, Nrp1 in surface representation and a schematic drawing (middle panel). b, Ribbon representation of Sema3A-PlxnA2-Nrp1 superposed onto Sema6A-PlxnA2 (Protein Data Bank (PDB) code 3OKY) based on the sema domains of a Sema3A and Sema6A molecule (left) and enlargement (right). c-e, Opened view. c, Interfaces of Sema3A-PlxnA2 (green), Sema3A-Nrp1 (red), Nrp1-PlxnA2 (blue) and Sema3A-Sema3A (yellow) and interface mutants used in biophysical and cellular assays (black). d, Mapping of previous interaction data: Sema3A mutations K108N, H216N and R404E (ruby), Sema3A derived peptide 363-380 (ref. 33) (purple), Nrp1 mutations (2AB, 2C, 3D, 2I) color-coded from most disrupting Sema3A binding (blue) to no effect (green) and a Nrp1 epitope (orange). e, Sema3A, PlxnA2 and Nrp1 color-coded according to residue conservation (from non-conserved, white, to conserved, black) based on alignments containing sequences from all vertebrate Sema3 and PlxnA class members and all Nrps. In b-e only the sema domains and Nrp1 domain a1 are shown.

Figure 3. The structure of Sema3A_S-P-I is very similar to that of Sema4D_S-P-I

a, Crystal structure of Sema3A_S-P-I in which the furin site (551RRTRR555) was mutated to R551A and R555A. The IG domain is only partially modelled. N-linked glycans are shown in grey stick representation. b, Superpositions of Sema3A_S-P-I with unliganded Sema3A_S (PDB code 1Q47) and Sema3A_S-F from the Sema3A-PlxnA2-Nrp1 complex described here. c, Superposition of Sema3A_S-P-I and SEMA4D_S-P-I (PDB code 1OLZ).

Figure 4. Interface mutants have reduced interaction and activity

a, Interface mutations (see also Fig. 2c and Supplementary Fig. 6) or sequestering of Ca²⁺ (by EDTA) reduce interactions between proteins. b, Mutant Sema3A has reduced collapse-inducing activity. Adult mouse DRG were incubated with Sema3A_S-P, Sema3A_S-PL353N P355S (both at 10 and 100 nM) or vehicle and scored for numbers of intact and collapsed growth cones. For each experimental condition at least 200 growth cones were used. Each data point is the mean ± s.e.m. of several independent experiments (6 for vehicle, 4 for 10 nM wt Sema3A, 3 for 100 nM wt Sema3A, 5 for 10 nM mutant Sema3A and 5 for 100 nM mutant Sema3A). (*p<0.05 by unpaired t-test). c, Representative images of non-collapsed (left panel) and Sema3A_S-P collapsed (right panel) DRG growth cones. Scale bar, 50 μm.

Figure 5. Model for secreted Sema3-PlxnA-Nrp and cell-surface bound Sema6-PlxnA signaling

Nrp and PlxnA can form cis complexes independent of Sema3 binding. Binding of Sema3 further stabilizes Nrp-PlxnA interaction and induces PlxnA2 dimerization. Nrp-Nrp interaction via the MAM domain^-, transmembrane helix or PlxnA-PlxnA interaction may seed further oligomerization (top panel). In absence of Nrp, PlxnA cannot signal via Sema3 but retains the ability to signal via membrane bound Sema6 (bottom panel).