Structural Principles in Robo Activation and Auto-inhibition

Reut Barak; Galit Yom-Tov; Julia Guez-Haddad; Lital Gasri-Plotnitsky; Roy Maimon; Moran Cohen-Berkman; Andrew A. McCarthy; Eran Perlson; Sivan Henis-Korenblit; Michail N. Isupov; Yarden Opatowsky

doi:10.1016/j.cell.2019.02.004

Structural Principles in Robo Activation and Auto-inhibition

Reut Barak
, Galit Yom-Tov
, Julia Guez-Haddad
, Lital Gasri-Plotnitsky
, Roy Maimon
, Moran Cohen-Berkman
, Andrew A. McCarthy
, Eran Perlson
, Sivan Henis-Korenblit
, Michail N. Isupov
, Yarden Opatowsky

Tel Aviv University
European Molecular Biology Laboratory
University of Exeter

Research output: Contribution to journal › Article › peer-review

40 Scopus citations

Abstract

Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos’ switch from “off” to “on” states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1–8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation. Intra- and inter-molecular contacts of Robo receptor auto-inhibit its dimerization, critical for axon guidance and signaling.

Original language	English
Pages (from-to)	272-285.e16
Journal	Cell
Volume	177
Issue number	2
DOIs	https://doi.org/10.1016/j.cell.2019.02.004
State	Published - 4 Apr 2019

Bibliographical note

Publisher Copyright:
© 2019 Elsevier Inc.

Funding

We thank Prof. Abdussalam Azem from Tel-Aviv University for AUC measurements and analysis. We thank Drs. Avi Jacob and Irit Shoval from the Bar Ilan University scientific equipment center for help in light microscopy image acquisition and ImageStream FACS analysis and Jennifer Israel Cohen Benichou for C. elegans data statistical analysis. We thank members of the Opatowsky and Henis-Korenblit laboratories for technical assistance, Dr. Einav Gross of the Hebrew University Faculty of Medicine and Dr. Moshe Dessau of Bar-Ilan University Faculty of Medicine for useful advice, Prof. Cornelia Bargmann of the Chan Zuckerberg Initiative for the gift of sax-3 template DNA, and Dr. David Sprinzak of Tel Aviv University for the gift of Notch and Delta expression vectors. Some nematode strains used in this work were provided by the Caenorhabditis Genetics Center. We thank the staff of the ESRF and EMBL-Grenoble ID30B, ID23, and ID29 of ESRF and the staff of BESSY II for assistance in using beamlines BL14.1 and 14.2. This work was supported by funds from ICRF (to Y.O.) and ISF (grant nos. 182/10 and 1425/15 ) (to Y.O.).

Funders	Funder number
Israel Cancer Research Fund
Israel Science Foundation	182/10, 1425/15

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10.1016/j.cell.2019.02.004

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title = "Structural Principles in Robo Activation and Auto-inhibition",

abstract = "Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos{\textquoteright} switch from “off” to “on” states remains unclear. Here, we report a 3.6 {\AA} crystal structure of the intact human Robo2 ectodomain (domains D1–8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation. Intra- and inter-molecular contacts of Robo receptor auto-inhibit its dimerization, critical for axon guidance and signaling.",

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AU - Cohen-Berkman, Moran

AU - McCarthy, Andrew A.

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AU - Isupov, Michail N.

AU - Opatowsky, Yarden

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AB - Proper brain function requires high-precision neuronal expansion and wiring, processes controlled by the transmembrane Roundabout (Robo) receptor family and their Slit ligands. Despite their great importance, the molecular mechanism by which Robos’ switch from “off” to “on” states remains unclear. Here, we report a 3.6 Å crystal structure of the intact human Robo2 ectodomain (domains D1–8). We demonstrate that Robo cis dimerization via D4 is conserved through hRobo1, 2, and 3 and the C. elegans homolog SAX-3 and is essential for SAX-3 function in vivo. The structure reveals two levels of auto-inhibition that prevent premature activation: (1) cis blocking of the D4 dimerization interface and (2) trans interactions between opposing Robo receptors that fasten the D4-blocked conformation. Complementary experiments in mouse primary neurons and C. elegans support the auto-inhibition model. These results suggest that Slit stimulation primarily drives the release of Robo auto-inhibition required for dimerization and activation. Intra- and inter-molecular contacts of Robo receptor auto-inhibit its dimerization, critical for axon guidance and signaling.

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Structural Principles in Robo Activation and Auto-inhibition

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