binding mode between LPHN3-OLF and FLRT3-LRR revealed by the crystallographic study an N-linked glycosylation site was introduced in the FLRT3-LRR interfacing surface and compared to a glycosylated mutant outside of the binding interface. At the interfacing position 160 we substituted Phe160 with an Asn, to complete the canonical Nx(S/T) sequon. As a control mutant, the surface residue Asn115 was glycosylated by substituting Arg117 to a Thr (Table 1). The additional N-linked glycosylation was verified by SDS-PAGE showing slower migrating bands for the Phe160Asn and Arg117Thr mutants, compared to the Phe160Ala or the wild-type construct (Figure 5D,E and Table 1). The glycosylated Arg117Thr mutant had a measured dissociation constant KD of 27nM, typical of wild type values (Figure 5F and Table 1). In contrast, Phe160Asn mutant showed no detectable binding in similar conditions (Figure 5G and Table 1), indicating that introducing bulky sugar moieties in the interface observed in the crystal structure, severely impacted the LPHN3-OLF/FLRT3-LRR association. As the Phe160 side chain was found to establish a direct interaction with the LPHN3-OLF Tyr245 in the complex structure, an Ala mutant was also designed to get insights into the importance of this specific position for the affinity between the two partners. For
