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An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly

Nature Structural & Molecular Biology volume 17, pages 398–402 (2010)Cite this article

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Abstract

Erythropoetin-producing hepatoma (Eph) receptors are cell-surface protein tyrosine kinases mediating cell-cell communication. Upon activation, they form signaling clusters. We report crystal structures of the full ectodomain of human EphA2 (eEphA2) both alone and in complex with the receptor-binding domain of the ligand ephrinA5 (ephrinA5 RBD). Unliganded eEphA2 forms linear arrays of staggered parallel receptors involving two patches of residues conserved across A-class Ephs. eEphA2–ephrinA5 RBD forms a more elaborate assembly, whose interfaces include the same conserved regions on eEphA2, but rearranged to accommodate ephrinA5 RBD. Cell-surface expression of mutant EphA2s showed that these interfaces are critical for localization at cell-cell contacts and activation-dependent degradation. Our results suggest a 'nucleation' mechanism whereby a limited number of ligand-receptor interactions 'seed' an arrangement of receptors which can propagate into extended signaling arrays.

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Figure 1: Crystal structures of the complete EphA2 extracellular region (eEphA2) alone and in complex with ephrinA5 RBD.
Figure 2: The eEphA2–ephrinA5 RBD complex: structural changes within eEphA2 and formation of array-like clusters.
Figure 3: EphA2 localization at cell-cell contacts depends on ectodomain clustering.
Figure 4: A mechanism for the nucleation and propagation of EphA2 signaling assemblies.

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Acknowledgements

We thank M. Walsh for assistance during data collection, B. Janssen for aiding with crystallographic data analysis, T.S. Walter for advice on protein crystallization, M. Jones and W. Lu for technical assistance, R. Gilbert for analytical ultracentrifugation measurements, the Molecular Cytogenetics and Microscopy Core facility of the Wellcome Trust Centre for Human Genetics, in particular K.J. Morris and N. Alsamhouri, for their help with confocal microscopy and D.I. Stuart for critical reading of the manuscript. This research was funded by Cancer Research UK. A.R.A. is funded by the UK Medical Research Council and E.Y.J. by Cancer Research UK. The work was supported by the Wellcome Trust Core Award, grant no. 075491/Z/04.

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Authors and Affiliations

  1. Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK

    Elena Seiradake, Karl Harlos, Geoff Sutton, A Radu Aricescu & E Yvonne Jones

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  1. Elena Seiradake
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Contributions

E.S. conducted crystallographic and cellular studies; K.H. performed crystal mounting and oversaw X-ray data collection; G.S. led the MALS analysis; A.R.A. and E.Y.J. participated in study design and oversaw all aspects of the work.

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Correspondence to A Radu Aricescu or E Yvonne Jones.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8, Supplementary Methods and Supplementary Table 1 (PDF 5250 kb)

Supplementary Video 1

Z-stack images of HEK293T cells expressing EphA2 as mVenus-fusion protein (green). EphA2 localizes at cell-cell contacts. (MOV 853 kb)

Supplementary Video 2

Z-stack images of HEK293T cells expressing the EphA2 interface D mutant L254D+V255D as mVenus fusion protein (green). Mutant EphA2 does not localize at cell-cell contacts. (MOV 654 kb)

Supplementary Video 3

Z-stack images of HEK293T cells expressing the EphA2 deletion construct eEphA2EphA2 as mVenus fusion protein (green). This construct lacks all C-terminal intracellular EphA2 domains. eEphA2EphA2 localizes at cell-cell contacts. (MOV 525 kb)

Supplementary Video 4

Z-stack images of HEK293T cells expressing the EphA2 deletion construct eEphA2Δe as mVenus fusion protein (green). This construct lacks the four N-terminal extracellular EphA2 domains LBD, sushi, EGF, FN1. eEphA2Δe does not localize at cell-cell contacts. (MOV 665 kb)

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Seiradake, E., Harlos, K., Sutton, G. et al. An extracellular steric seeding mechanism for Eph-ephrin signaling platform assembly. Nat Struct Mol Biol 17, 398–402 (2010). https://doi.org/10.1038/nsmb.1782

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