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Structural basis for semaphorin signalling through the plexin receptor

Abstract

Semaphorins and their receptor plexins constitute a pleiotropic cell-signalling system that is used in a wide variety of biological processes, and both protein families have been implicated in numerous human diseases1,2,3,4. The binding of soluble or membrane-anchored semaphorins to the membrane-distal region of the plexin ectodomain activates plexin’s intrinsic GTPase-activating protein (GAP) at the cytoplasmic region, ultimately modulating cellular adhesion behaviour5. However, the structural mechanism underlying the receptor activation remains largely unknown. Here we report the crystal structures of the semaphorin 6A (Sema6A) receptor-binding fragment and the plexin A2 (PlxnA2) ligand-binding fragment in both their pre-signalling (that is, before binding) and signalling (after complex formation) states. Before binding, the Sema6A ectodomain was in the expected ‘face-to-face’ homodimer arrangement, similar to that adopted by Sema3A and Sema4D, whereas PlxnA2 was in an unexpected ‘head-on’ homodimer arrangement. In contrast, the structure of the Sema6A–PlxnA2 signalling complex revealed a 2:2 heterotetramer in which the two PlxnA2 monomers dissociated from one another and docked onto the top face of the Sema6A homodimer using the same interface as the head-on homodimer, indicating that plexins undergo ‘partner exchange’. Cell-based activity measurements using mutant ligands/receptors confirmed that the Sema6A face-to-face dimer arrangement is physiologically relevant and is maintained throughout signalling events. Thus, homodimer-to-heterodimer transitions of cell-surface plexin that result in a specific orientation of its molecular axis relative to the membrane may constitute the structural mechanism by which the ligand-binding ‘signal’ is transmitted to the cytoplasmic region, inducing GAP domain rearrangements and activation.

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Figure 1: Crystal structure of Sema6A and PlxnA2 ectodomain fragments in pre-signalling and post-signalling states.
Figure 2: Authenticity of the semaphorin–plexin interface is confirmed by mutational experiments.
Figure 3: The Sema6A face-to-face homodimer represents a signalling-competent active conformation.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The coordinates of Sema6ASP, PlxnA2SP and their complex have been deposited in the Protein Data Bank under accession codes 3AFC, 3AL9 and 3AL8, respectively.

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Acknowledgements

We would like to thank Y. Yamada, N. Matsugaki and N. Igarashi of Photon Factory and Y. Kawano and N. Shimizu of SPring-8 BL-41XU for their help with the X-ray data collection; A. Rowe for discussions on the sedimentation equilibrium data analysis; C. Wu for performing the Sema6A–AP binding assay; K. Tamura-Kawakami and M. Nampo for their technical support; and M. Nakano for preparation of the manuscript. This work was supported in part by a ‘Target Proteins Research Program (TPRP)’ grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).

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T.N. and J.T. conceived the project. No.Y. and E.M. expressed, purified and crystallized the proteins. Y.M., Na.Y. and T.T. performed cell biological assays. M.N. and S.U. performed analytical ultracentrifugation experiments. T.N. and No.Y. collected X-ray diffraction data. T.N. and J.T. determined and analysed the structures. T.N., S.U., Y.G., A.K. and J.T. wrote the paper.

Corresponding author

Correspondence to Junichi Takagi.

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The authors declare no competing financial interests.

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Nogi, T., Yasui, N., Mihara, E. et al. Structural basis for semaphorin signalling through the plexin receptor. Nature 467, 1123–1127 (2010). https://doi.org/10.1038/nature09473

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