To test how the stabilization of the 316–329 loop affects the LPHN3-OLF/FLRT3-LRR association, we engineered an Asp332Ala and a Tyr323Ala that should disrupt the Ca2+-driven stabilization of this loop, and measured their interaction with FLRT3-LRR. All mutant proteins were first analyzed by SEC to reveal potential misfolding or oligomerization state abnormalities. As expected, the expression level of all mutants and their elution profile were identical to the ones measured for the WT proteins (data not shown). The mutant Asp332Ala showed a decreased affinity (KD ~170 nM) (Table 1), indicating that Asp332 is important by strengthening the closed state through the coordination of Ca2+ and the Asp332-Tyr323 hydrogen bond. Furthermore, the mutant Tyr323Ala showed no detectable binding in conditions similar to the wild type measurements, which strongly suggests that Tyr323 is key to maintain the “closed” state. Moreover, to confirm the 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
