PDBsum entry 2vr9

Cell adhesion

PDB id: 2vr9

Contents

Protein chains

199 a.a. *

* Residue conservation analysis

PDB id:

2vr9

Name:

Cell adhesion

Title:

Drosophila robo ig1-2 (tetragonal form)

Structure:

Roundabout 1. Chain: a, b, c. Fragment: ig1-2, residues 51-254. Synonym: robo. Engineered: yes

Source:

Drosophila melanogaster. Fruit fly. Organism_taxid: 7227. Expressed in: homo sapiens. Expression_system_taxid: 9606. Expression_system_cell_line: 293-ebna.

Resolution:

3.20Å R-factor: 0.237 R-free: 0.274

Authors:

N.Fukuhara,J.A.Howitt,S.Hussain,E.Hohenester

Key ref:

N.Fukuhara et al. (2008). Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo. J Biol Chem, 283, 16226-16234. PubMed id: 18359766 DOI: 10.1074/jbc.M800688200

Date:

28-Mar-08 Release date: 08-Apr-08

Protein chains

O44924  (O44924_DROME) -  Roundabout 1 from Drosophila melanogaster

Seq: 1395 a.a.

Struc: 199 a.a.

DOI no: 10.1074/jbc.M800688200

J Biol Chem 283:16226-16234 (2008)

PubMed id: 18359766

Structural and functional analysis of slit and heparin binding to immunoglobulin-like domains 1 and 2 of Drosophila Robo.

N.Fukuhara, J.A.Howitt, S.A.Hussain, E.Hohenester.

ABSTRACT

Recognition of the secreted protein Slit by transmembrane receptors of the Robo family provides important signals in the development of the nervous system and other organs, as well as in tumor metastasis and angiogenesis. Heparan sulfate (HS) proteoglycans serve as essential co-receptors in Slit-Robo signaling. Previous studies have shown that the second leucinerich repeat domain of Slit, D2, binds to the N-terminal immunoglobulin-like domains of Robo, IG1-2. Here we present two crystal structures of Drosophila Robo IG1-2, one of which contains a bound heparin-derived oligosaccharide. Using structure-based mutagenesis of a Robo IG1-5 construct we identified key Slit binding residues (Thr-74, Phe-114, Arg-117) forming a conserved patch on the surface of IG1; mutation of similarly conserved residues in IG2 had no effect on Slit binding. Mutation of conserved basic residues in IG1 (Lys-69, Arg-117, Lys-122, Lys-123), but not in IG2, reduced binding of Robo IG1-5 to heparin, in full agreement with the Robo-heparin co-crystal structure. Our collective results, together with a recent crystal structure of a minimal human Slit-Robo complex ( Morlot, C., Thielens, N. M., Ravelli, R. B., Hemrika, W., Romijn, R. A., Gros, P., Cusack, S., and McCarthy, A. A. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 14923-14928 ), reveal a contiguous HS/heparin binding surface extending across the Slit-Robo interface. Based on the size of this composite binding site, we predict that at least five HS disaccharide units are required to support Slit-Robo signaling.

Selected figure(s)

Figure 5.
FIGURE 5. Heparin oligosaccharide bound to Robo IG1-2. A, cartoon representation of the crystallographically independent Robo IG1-2 molecules A and B in the monoclinic crystal form. IG1 and IG2 are shown in orange and green, respectively. The bound heparin oligosaccharide is shown in ball-and-stick representation, with carbon atoms colored cyan. The C atoms of basic residues implicated in heparin binding are indicated by blue spheres. The side chains of Lys-69 and Arg-117 are defined by the electron density, whereas those of Lys-121 and Lys-123 are disordered (see text). B, another view of the bound heparin oligosaccharide, in an orientation roughly perpendicular to that of panel A. Heparin binding residues are labeled. C, stereo view of an unbiased simulated annealing omit map contoured at 2.4 , with the final model of the heparin tetrasaccharide superimposed on the map. D, superposition of the heparin oligosaccharides observed in different protein-heparin co-crystal structures: red, Robo IG1-2 (this study); purple, FGF2-FGFR1 complex (33); green, FGF1-FGFR2 complex (31); pink, hepatocyte growth factor/scatter factor NK1 fragment (36).

Figure 6.
FIGURE 6. Composite heparin binding site in the Slit-Robo complex. Shown is a model of the human Slit2 D2-Robo1 IG1-2 complex, obtained by substituting Robo1 IG1 with crystal form 1 of Robo1 IG1-2 (14). A, surface representation: pale brown, Robo1 IG1; pale green, Robo1 IG2; pale pink, Slit2 D2. B, electrostatic surface representation of the same complex (red, negative potential; blue, positive potential). The positions of heparin binding residues in Slit D2 (21) and Robo IG1-2 (this study) are labeled. Human Robo1 residues Lys-81, Arg-131, Arg-136, and Lys-137 correspond, respectively, to Drosophila Robo residues Lys-69, Arg-117, Lys-122, and Lys-123. The location of the bound heparin oligosaccharide in the Robo IG1-2 structure is indicated by a semitransparent circle. A heparin octasaccharide (dp8) and decasaccharide (dp10) are drawn to scale and shown in ball-and-stick representation. HS/heparin is suggested to bind to the extended basic surface across the Slit-Robo interface (see text).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2008, 283, 16226-16234) copyright 2008.

Figures were selected by an automated process.