, 2009 and Yamagishi et al., 2011), although the absolute values are lower, presumably due to differences in the techniques applied. We also used SPR to test the binding of FLRT2LRR to Unc5D fragments encompassing different regions of the ectodomain BMS-354825 order (Unc5Decto, Unc5DIg12, Unc5DIg1, Unc5DIg2, and Unc5DT12; depicted in Figure 1A). The results showed that the N-terminal Unc5D Ig domain (Unc5DIg1) harbors the major FLRT2LRR-binding site (Figure 1C). We determined the crystal structures of mouse FLRT2LRR and FLRT3LRR. Crystallographic details are provided in Table S2. Both structures consist of ten lrr repeats plus flanking

cap structures, together forming a horseshoe-shaped solenoid ( Figures 1D–1F, S1A, and S1B). Superposition underscores the similarity of the two structures with a root-mean-square Nintedanib in vivo deviation (rmsd) ( Krissinel and Henrick, 2004) of 1.17 Å for 320 (out of 321) corresponding Cα atoms. We generated sequence conservation scores ( Glaser et al., 2003) using alignments of FLRT2 and FLRT3 from mouse, chicken, frog, and fish and mapped these onto the FLRTLRR structures. A sequence-conserved patch extends from the concave to a lateral side surface of both FLRTLRR structures

( Figures 1G and S1B). Comparison of FLRT2LRR with structures in the Dali database ( Holm and Rosenström, 2010) shows strongest similarity (rmsd for 264 aligned Cα atoms = 1.8) with the cell adhesion protein decorin,

which is known to dimerize via the concave surface of its LRR domain ( Scott et al., 2004). The predominantly charged concave surfaces of FLRT2LRR and FLRT3LRR ( Figures 1H and S1B) provide lattice contacts in all of our crystal structures ( Figure S1), suggesting that these regions could mediate functional FLRT-FLRT interactions. We determined the crystal structure of only rat Unc5DIg1 (Table S2). The domain conforms to the Ig subtype 1 topology (Chothia and Jones, 1997) (Figure 2A). The structure is most similar to that of the N-terminal Ig domain of receptor protein tyrosine phosphatase delta (RPTPδ, rmsd for 86 aligned Cα atoms = 1.9 Å), although Unc5D lacks the positively charged surface patch that mediates the RPTPδ-glycosaminoglycan interaction (Coles et al., 2011). We also solved a crystal structure for Unc5AIg12T2 (Table S2), thereby revealing the fold of the second Ig domain, also subtype 1, and the TSP domain (Figure 2B). The crystallized construct corresponds to the complete human Unc5A isoform 1 ectodomain. The overall structure is elongated and lacks extended interdomain linkers. All human Unc5A isoforms and mouse Unc5A isoform 2 lack the first of the two TSP domains that are present in other Unc5 homologs. Otherwise, the sequences of Unc5A–D are 44%–63% conserved between the human Unc5 homologs. We solved the crystal structure of FLRT2LRR in complex with Unc5DIg1 (Table S2).