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. 2021 May 26;12(1):3172.
doi: 10.1038/s41467-021-23541-x.

Architecture of the Sema3A/PlexinA4/Neuropilin tripartite complex

Defen Lu #  1 , Guijun Shang #  1 , Xiaojing He #  2 , Xiao-Chen Bai  3   4 , Xuewu Zhang  5   6
Affiliations

Architecture of the Sema3A/PlexinA4/Neuropilin tripartite complex

Defen Lu et al. Nat Commun. .

Abstract

Secreted class 3 semaphorins (Sema3s) form tripartite complexes with the plexin receptor and neuropilin coreceptor, which are both transmembrane proteins that together mediate semaphorin signal for neuronal axon guidance and other processes. Despite extensive investigations, the overall architecture of and the molecular interactions in the Sema3/plexin/neuropilin complex are incompletely understood. Here we present the cryo-EM structure of a near intact extracellular region complex of Sema3A, PlexinA4 and Neuropilin 1 (Nrp1) at 3.7 Å resolution. The structure shows a large symmetric 2:2:2 assembly in which each subunit makes multiple interactions with others. The two PlexinA4 molecules in the complex do not interact directly, but their membrane proximal regions are close to each other and poised to promote the formation of the intracellular active dimer for signaling. The structure reveals a previously unknown interface between the a2b1b2 module in Nrp1 and the Sema domain of Sema3A. This interaction places the a2b1b2 module at the top of the complex, far away from the plasma membrane where the transmembrane regions of Nrp1 and PlexinA4 embed. As a result, the region following the a2b1b2 module in Nrp1 must span a large distance to allow the connection to the transmembrane region, suggesting an essential role for the long non-conserved linkers and the MAM domain in neuropilin in the semaphorin/plexin/neuropilin complex.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Overall structure of the Sema3A/PlexinA4/Nrp1 complex.
a Domain structures of mouse Sema3A, PlexinA4, and Nrp1. Colored parts indicate boundaries of constructs used in the structural analyses. b Overview of the cryo-EM map of the Sema3A/PlexinA4/Nrp1 complex. The color scheme is the same as a. c Atomic model of the Sema3A/PlexinA4/Nrp1 complex based the cryo-EM reconstruction.
Fig. 2
Fig. 2. Importance of the linker regions in the formation of the Sema3A/PlexinA4/Nrp1 complex.
a Roles of the linkers in connecting various parts of the proteins in the tripartite complex on the cell surface. The crystal structure of the MAM domain of Nrp1 (PDB ID: 5l73) is docked between the a2b1b2 module and the plasma membrane. Thick lines represent the linker regions. The arrows in the right panel (top view) indicate that the MAM-linker region may use two alternative paths (through the center or outside of the PlexinA4 ring) to connect to the TM region of Nrp1. b Sequence alignment of the b2-MAM and MAM-TM linkers from mouse Nrp1 and Nrp2. c Sequence alignment of the tail regions of mouse class 3 semaphorins. Putative furin-cleavage sites are highlighted.
Fig. 3
Fig. 3. Interface between PlexinA4 and Nrp1-a1.
a Overview of the PlexinA4-Sema/Nrp1-a1 interface. For clarity, only one Nrp1-a1 domain and one PlexinA4 molecule from the 2:2:2 complex are shown. One complex of PlexinA2/Nrp1-a1 from PDB ID 4gza is superimposed for comparison. b Detailed view of the PlexinA4-Sema/Nrp1-a1 interface. c Sequence alignment of the loop region which in mouse PlexinA4 contains Lys343 that is important for interacting with Nrp1-a1.
Fig. 4
Fig. 4. Interfaces between Sema3A and Nrp1.
a Overview of the Sema3A/ Nrp1 interfaces. For clarity, only one Nrp1 molecule and the Sema3A dimer from the 2:2:2 complex are shown. One complex of Sema3A/Nrp1-a1 from PDB ID 4gza is superimposed for comparison. b Detailed view of the interface between the Sema3A-Sema domain and the Nrp1-a1 domain. c Detailed view of the interface between the Sema3A-Sema domain and the Nrp1-a2 domain. d Sequence alignment of the loop region which in mouse Sema3A contains Lys497 that is important for interacting with Nrp1-a2.
Fig. 5
Fig. 5. COS7 cell collapse assays for mutants of Sema3A, PlexinA4, and Nrp1.
a Images of COS7 cell collapse. Cells stably expressing various combinations of PlexinA4 and Nrp1 constructs were treated with different Sema3A constructs at 5 nM for 30 min. Cells are visualized by immuno-staining for myc-tagged PlexinA4 (green). Nuclei are stained with DAPI (blue). One representative image from at least three biological repeats is shown for each group. b Quantification of cell collapse. The individual data points, mean, and s.e.m of the percentage of cell collapse from at least three biological repeats are shown (n = 4 for Sema3A-WT/PlexinA4-WT/Nrp1-WT, n = 5 for Sema3A-WT/PlexinA4-WT/Nrp1-S251E, n = 6 for Sema3A-WT/PlexinA4-WT/Nrp1-A252E, n = 3 for other groups). Statistical significance p-values between wild-type and each mutant group were determined by two-tailed Student’s t-test and shown on the top of the bars. Scale bar, 50 µm.
Fig. 6
Fig. 6. Cartoon model of the regulatory mechanisms of class A plexins.
Prior to ligand binding, class A plexins form the inhibitory dimer that prevents the formation of the intracellular active dimer (left panel). It is possible that neuropilin uses its a1 domain to bind plexin, disrupting the inhibitory dimer and priming plexin for activation (lower panel). Neuropilin could bind the semaphorin dimer on its own, considering the strong interaction between the two (upper panel). Ultimately, the semaphorin dimer induces the formation of the 2:2:2 complex, which in turn induces the intracellular active dimer of plexin for signaling (right panel). Whether and how the transmembrane region of plexin and neuropilin interact in the 2:2:2 complex is unclear.
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