Robo Ig4 Is a Dimerization Domain

Yom-Tov, Galit; Barak, Reut; Matalon, Omri; Barda‐Saad, Mira; Guez-Haddad, Julia; Opatowsky, Yarden

doi:10.1016/j.jmb.2017.10.002

Cited by 19 publications

(16 citation statements)

References 45 publications

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“…Preclusion of D4 Dimerization Interface in the hRobo2 D1-8 Structure D4 is located at the center of the hRobo2 D1-8 structure, flanked by D3 on its N 0 terminal side, D5 and D6 on one domain face, and by D7 on the other ( Figures 1B and 2C). Remarkably, we have previously identified the same surface of D4, which is sequestered by D7 as a direct mediator of dimeric hRobo2 interactions (Yom-Tov et al, 2017). Specifically, we showed that a predominantly hydrophobic surface on D4 mediates close homotypic contacts with a reciprocal D4 in the crystal lattice of hRobo2 D4-5 (PDB: 5NOI) (Figures 2A, S1, and S3).…”

Section: Overall Structure Of An Intact Hrobo2 Ectodomain Monomersupporting

confidence: 58%

“…These structures pro- vided important information about Robo binding to Slit and HSPGs and hRobo1 ectodomain proteolysis. More recently, our crystal structure of hRobo2 D4-5 (PDB: 5NOI) (Yom-Tov et al, 2017) identified D4 as a dimerization domain in vitro, and the crystal structures of hRobo1 D1-4 (PDB: 5OPE, 5O5G) that, as will be discussed here, reveals an identical dimerization interface. Also insightful is the low-resolution electron microscopy single particle structure of hRobo1 D1-8 (Aleksandrova et al, 2018), showing a head-to-head dimer of dimers trans arrangement of the receptor.…”

Section: Introductionmentioning

confidence: 70%

“…The identification of D4 as a dimerization domain of hRobo2 in vitro and cell-size assays (Yom-Tov et al, 2017), together with identical D4 dimerization interfaces observed in hRobo2 D4-5 (PDB: 5NOI) and hRobo1 D1-4 (PDB: 5OPE, 5O5G) (Figures 2 and S3), have further focused attention on Robo D4mediated dimerization. A high level of evolutionary sequence conservation is observed in the D4 dimerization interface (Yom-Tov et al, 2017), including those of hRobo3 and the C. elegans SAX-3 ( Figure S1). Here, we further demonstrate homo-dimerization of hRobo1, 2, 3 and SAX-3 using a Robo-cKIT chimera assay (Figure 3).…”

Section: D4-mediated Dimerization Is Important For Robo Signalingmentioning

confidence: 87%

“…The D4-D4 dimerization interface, as revealed in the crystal structures of hRobo2 D4-5 and hRobo1 D1-4 , includes several conserved residues, one of which is hRobo2 F357 / hRobo1 F394 (Figures S1 and S3). Previously, we found that a F357R substitution eliminates hRobo2 D4-5 dimer formation in AUC measurements (Yom-Tov et al, 2017) and, therefore, targeted the homologous SAX-3 F360 in the C. elegans AVM guidance model. As anticipated, the Pmec-7::sax-3 F360R transgene showed a loss-of-function phenotype, with only 30% of the total animals (n = 91) with a ''turn'' phenotype, 53% ''straight,'' and 16% other phenotypes.…”

Section: Importance Of D4-mediated Dimerization In Vivomentioning

confidence: 99%

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Structural Principles in Robo Activation and Auto-inhibition

Barak

Yom-Tov

Guez-Haddad

et al. 2019

Cell

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Graphical Abstract Highlights d Crystal structure of the intact human Robo2 ectodomain at 3.6 Å d Dimerization through domain 4 (D4) is required for Robo axon guidance d Robo receptors have an auto-inhibited conformation in which D4 is blocked d Slit may relieve Robo auto-inhibition, followed by dimerization and signaling SUMMARYProper 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.

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Section: Overall Structure Of An Intact Hrobo2 Ectodomain Monomersupporting

confidence: 58%

Section: Introductionmentioning

confidence: 70%

Section: D4-mediated Dimerization Is Important For Robo Signalingmentioning

confidence: 87%

Section: Importance Of D4-mediated Dimerization In Vivomentioning

confidence: 99%

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Structural Principles in Robo Activation and Auto-inhibition

Barak

Yom-Tov

Guez-Haddad

et al. 2019

Cell

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“…Robo family members have been shown previously to interact homomerically and exist predominantly as homodimers (22,23). Additionally, the fourth Ig (Ig4) domain of Robo2 was identified as a dimerization domain (24). NELL proteins did not bind to intact Robo2 and its deletion mutants containing the Ig4 domain ( Fig.…”

Section: Amino Acid Substitution At the Position Of The Predicted Dimmentioning

confidence: 90%

Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2

Yamamoto

Kashiwagi

Ishihara

et al. 2019

Journal of Biological Chemistry

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The signaling pathways that are mediated by Slit ligands and their Roundabout (Robo) family of receptors play multifunctional roles in the development of the nervous system and other organs. A recent study identified neural epidermal growth factor-like (NEL)-like 2 (NELL2) as a novel ligand for Robo3. In this study, we carried out a comprehensive analysis of the interaction between NELL1 and the Robo family of receptors and demonstrated that Robo2 contains a cryptic binding site for both NELL1 and NELL2. NELL1/2 binds to the first fibronectin type III (FNIII) domain of Robo2 but not to intact Robo2. Mutation analysis revealed that several amino acids within the first FNIII domain are critical for NELL1 binding to Robo2 but not to Robo1. The Robo2 deletion mutants without the fourth immunoglobulin domain and single amino acid substitution mutants that can influence the architecture of the ectodomain facilitated binding to NELL1/2. Acidic conditions increased the binding affinity of Robo2 for NELL1. These results suggest that Robo2 functions as a receptor for NELL1/2, particularly under circumstances where Robo2 undergoes proteolytic digestion. If this is not the case, conformational changes of the ectodomain of Robo2 may unmask the binding site for NELL1/2.

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Roles of Slit Ligands and Their Roundabout (Robo) Family of Receptors in Bone Remodeling

Niimi

2020

Advances in Experimental Medicine and Biology

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Robo Ig4 Is a Dimerization Domain

Cited by 19 publications

References 45 publications

Structural Principles in Robo Activation and Auto-inhibition

Structural Principles in Robo Activation and Auto-inhibition

Robo2 contains a cryptic binding site for neural EGFL-like (NELL) protein 1/2

Roles of Slit Ligands and Their Roundabout (Robo) Family of Receptors in Bone Remodeling

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