Structural insights into the Slit-Robo complex

Abstract

Slits are large multidomain leucine-rich repeat (LRR)-containing proteins that provide crucial guidance cues in neuronal and vascular development. More recently, Slits have been implicated in heart morphogenesis, angiogenesis, and tumor metastasis. Slits are ligands for the Robo (Roundabout) receptors, which belong to the Ig superfamily of transmembrane signaling molecules. The Slit-Robo interaction is mediated by the second LRR domain of Slit and the two N-terminal Ig domains of Robo, but the molecular details of this interaction and how it induces signaling remain unclear. Here we describe the crystal structures of the second LRR domain of human Slit2 (Slit2 D2), the first two Ig domains of its receptor Robo1 (Ig1-2), and the minimal complex between these proteins (Slit2 D2-Robo1 Ig1). Slit2 D2 binds with its concave surface to the side of Ig1 with electrostatic and hydrophobic contact regions mediated by residues that are conserved in other family members. Surface plasmon resonance experiments and a mutational analysis of the interface confirm that Ig1 is the primary domain for binding Slit2. These structures provide molecular insight into Slit-Robo complex formation and will be important for the development of novel cancer therapeutics.

Fig. 1.

Structure of human Robo1 Ig1–2. (A) Ribbon diagram. The disulfide bridges are in yellow, and the box indicates the region highlighted in B. (B) Residues involved in interdomain contacts of the Ig1-Ig2 interface. (C) Ribbon diagram of the two Ig1–2 crystal forms showing the hinge movement of Ig2. (D) Sequence alignment of Ig1 domains of human Robo1, -2, -3, and -4 and of the Ig2 domain of human Robo1. Residue numbering is for Robo1 Ig1 (above) and Robo1 Ig2 (below). Slit2 D2-binding residues selected for mutagenesis are marked with a star, and residues strictly conserved between the Ig1 domain of human Robo1, -2, -3, and -4 are shown in red.

Fig. 2.

Structure of Slit2 D2 bound to Robo1 Ig1. Ig1 is in green; Slit2 D2 N- and C-terminal caps are in purple and blue, respectively; LRRs 1–6 are in orange; and the disulfide bridges are in yellow. Interacting residues are shown in stick representation. (A) Ribbon diagram of the complex in two orthogonal orientations. (B) Electrostatic region of the Slit2 D2-Ig1 interface. (C) Hydrophobic region of the Slit2 D2-Ig1 interface. (D) Relative Slit2 D2-binding capacity of Robo1 Ig1 variants. Results are expressed as maximal Slit2 D2-binding, normalized with respect to the maximal Slit2 D2-binding capacity of wild-type Robo1 Ig1.

Fig. 3.

Flexibility of the Robo1 Ig1-Ig2 linkage. (A) Superposition of the two Robo1 Ig1–2 crystal forms with the structure of Slit2 D2 bound to Robo1 Ig1, in two orientations. Ig1 is in green; Ig2 is in cyan or violet; Slit2 D2 N- and C-terminal caps are in purple and blue, respectively; LRRs 1–6 are in orange; and the disulfide bridges are in yellow. Slit2 D2 residues involved in heparin binding are in red and are shown in stick representation. (B) Schematic of the Slit2-Robo1 domain organization with the flexible linkage marked by a curved arrow. The Robo1 Ig1 domain is shown in green, and the Slit2 D2 is in orange. All other domains are opaque, the Robo1 Ig2 is in magenta, the other Ig domains are in green, and the FN3 domains are in blue. The Slit2 LRR domains are colored in orange, and the EGF domains are in blue.