Binding site for Robo receptors revealed by dissection of the leucine-rich repeat region of Slit

Recognition of the large secreted protein Slit by receptors of the Robo family provides fundamental signals in axon guidance and other developmental processes. In Drosophila, Slit–Robo signalling regulates midline crossing and the lateral position of longitudinal axon tracts. We report the functional dissection of Drosophila Slit, using structure analysis, site-directed mutagenesis and in vitro assays. The N-terminal region of Slit consists of a tandem array of four independently folded leucine-rich repeat (LRR) domains, connected by disulphide-tethered linkers. All three Drosophila Robos were found to compete for a single highly conserved site on the concave face of the second LRR domain of Slit. We also found that this domain is sufficient for biological activity in a chemotaxis assay. Other Slit activities may require Slit dimerisation mediated by the fourth LRR domain. Our results show that a small portion of Slit is able to induce Robo signalling and indicate that the distinct functions of Drosophila Robos are encoded in their divergent cytosolic domains

Crystal Structure of the LG1-3 Region of the Laminin α2 Chain*

Laminins are large heterotrimeric glycoproteins with many essential functions in basement membrane assembly and function. Cell adhesion to laminins is mediated by a tandem of five laminin G-like (LG) domains at the C terminus of the α chain. Integrin binding requires an intact LG1-3 region, as well as contributions from the coiled coil formed by the α, β, and γ chains. We have determined the crystal structure at 2.8-Å resolution of the LG1-3 region of the laminin α2 chain (α2LG1-3). The three LG domains adopt typical β-sandwich folds, with canonical calcium binding sites in LG1 and LG2. LG2 and LG3 interact through a substantial interface, but LG1 is completely dissociated from the LG2-3 pair. We suggest that the missing γ chain tail may be required to stabilize the interaction between LG1 and LG2-3 in the biologically active conformation. A global analysis of N-linked glycosylation sites shows that the β-sandwich faces of LG1 are free of carbohydrate modifications in all five laminin α chains, suggesting that these surfaces may harbor the integrin binding site. The α2LG1-3 structure provides the first atomic view of the integrin binding region of laminins