Structure and functional relevance of the Slit2 homodimerization domain

Abstract

Slit proteins are secreted ligands that interact with the Roundabout (Robo) receptors to provide important guidance cues in neuronal and vascular development. Slit-Robo signalling is mediated by an interaction between the second Slit domain and the first Robo domain, as well as being dependent on heparan sulphate. In an effort to understand the role of the other Slit domains in signalling, we determined the crystal structure of the fourth Slit2 domain (D4) and examined the effects of various Slit2 constructs on chick retinal ganglion cell axons. Slit2 D4 forms a homodimer using the conserved residues on its concave face, and can also bind to heparan sulphate. We observed that Slit2 D4 frequently results in growth cones with collapsed lamellipodia and that this effect can be inhibited by exogenously added heparan sulphate. Our results show that Slit2 D4-heparan sulphate binding contributes to a Slit-Robo signalling mechanism more intricate than previously thought.

Figure 1

Structure of Slit2 D4 and the Decorin dimer in similar orientations. The Slit2 D4 and decorin amino-terminal caps are coloured purple, whereas their carboxy-terminal caps are coloured blue and cyan. The LRRs are coloured orange and the disulphide bridges are in yellow (Slit2 D4 N-terminal cap: Cys727–Cys733 and Cys731–Cys740; Slit2 D4 C-terminal cap: Cys863–Cys886 and Cys865–Cys907). The sulphate ion is indicated by sticks. (A) Human Slit2 D4 monomer. (B) Human Slit2 D4 dimer. (C) Bovine decorin monomer. (D) Bovine decorin dimer.

Figure 2

Molecular interactions at the dimer interface. (A) Central region of the Slit2 D4 dimerization interface. Residues that differ in chains (A) and (B) are shown as sticks. The β-switch loop of chain (B) is coloured purple. (B) The asymmetric dimer region with the chain (A) β-switch loop is coloured purple. (C) Chain (A) and (B) superpositions. The orientation of the ribbon and sticks model is as in (B) (chain (A) on top, chain (B) at the bottom). Lines represent the alternative residue conformation in the opposite chains, as seen in (A). (D) The β-switch loop of chain (A) and interacting residues. (E) The central four-histidine cluster and water network. (F) The sulphate binding site.

Figure 3

Sequence conservation and electrostatic surface representation of Slit2 D4 with the sulphate ion in sticks. (A) Structural representation, in four orientations and coloured as in Fig 1. (B) Sequence surface conservation as viewed in (A) and coloured in green: identical; yellow: partly conserved; white: poorly conserved. (C) Electrostatic surface potential calculated with Adaptive Poisson–Boltzmann Solver (APBS; Baker et al, 2001; from −2 coloured in red: negatively charged, to +2 coloured in blue: positively charged).

Figure 4

Biological activity of Slit2 contructs. (A) Stripe assay with axons given the choice between substrate-bound lanes covered with Slit2 D2, Slit2 D4 and FC compared with lanes covered with FC (indicated in magenta). Scale bar, 200 μm. (B) Representative single growth cones after treatment with soluble Slit2 D2 (full collapse), Slit2 D4 (bare branch morphology) and control treatment (intact morphology). Scale bar, 20 μm. (C) Heparan sulphate (HS) oligomers (12-mers) alter the effect of Slit2 D2 and D4 on growth cones incubated for 15 min with 100 μg/ml HS oligomers, followed by incubation for 30 min with 100 μg/ml HS oligomers and 50 μg/ml Slit2 D2 or D4, respectively. Blue bars indicate the percentage of intact growth cones, red bars indicate growth cones with bare branch morphology and green bars indicate fully collapsed growth cones. Bars indicate the s.e.m.; n indicates the number of growth cones scored.