Unc5Ig1 binds to FLRT2LRR burying a total of ∼1,280 Å2 protein surface, which is highly sequence conserved on both sides (Figures 2C and 2D). Superposition of Unc5AIg12T with Unc5DIg1 as found in complex with FLRT2LRR generates a model in which the domains downstream of Unc5 Ig1 extend away from the interface with FLRTLRR, suggesting that the Ig1 domain is the only interacting domain (Figure 2E). Based on this model alone, we cannot rule out that the extracellular FLRT regions downstream of the LRR domain also interact with Unc5. However, in SPR experiments we measured similar Unc5-binding affinities for FLRTecto and

FLRTLRR constructs (data not shown), suggesting that there is no major second Unc5-binding site on FLRT. We provide further support for this conclusion using a mutagenesis AZD9291 clinical trial approach (see

next section). The core of the FLRT2-Unc5D-binding interface contains predominantly hydrophobic and positively charged residues (Figures 2F and 2G). The conserved FLRT2 histidine H170 forms a central anchor point that reaches deep into a hydrophobic pocket formed by Unc5D F82, K84, W89, V135, W137, Dasatinib cost and K144 and likely provides a hydrogen bond to Unc5D W137 (Figure 2G). FLRT2 R191 and L215 may stabilize this arrangement by providing additional contacts to Unc5D F82 and W137. The main residues forming the hydrophobic FLRT2-binding surface of Unc5D are fully conserved in Unc5B (Figure 2H), with the exception of F82, which is replaced by a tyrosine (Y78). The high the degree of sequence conservation at the FLRT-Unc5-binding interface is in agreement with the observed binding promiscuity. Subtle differences in binding affinities

for different homologs are likely due to sequence variations at the periphery of the binding interface (Figure 2I). Histidine residues have a side chain pKa(His) of ∼6, below which they are protonated. We predicted that the protonated FLRT2 H170 would be incompatible with binding to the hydrophobic binding pocket on Unc5D. Indeed, at pH ∼5.7, Unc5Decto does not interact with FLRT2ecto (Figure S2A). Based on the crystal structures, we designed mutations in the FLRT2-Unc5D interface to disrupt binding. In FLRT2 H170E and H170N, we replaced the central histidine with a negative-charged residue or an N-linked glycosylation site, respectively. Neither of these mutants binds Unc5D in our assays, confirming the binding site we describe is essential for the interaction (Figure 3A). Also, the Unc5D mutants E88A+W89A+H91A and W89N+H91T show poor binding to FLRT2 (Figure 3A). Binding was unaffected by FLRT2 and Unc5D mutations at sites involved in minor interactions in the crystal (FLRT2 D248N+P250T, Unc5D L101N+E103T), suggesting that these sites are not physiologically relevant (Figure 3A). For subsequent functional analysis we chose the non-Unc5-binding FLRT2 mutant H170N and the non-FLRT2-binding Unc5D mutant W89N+H91T.