Crystal structure of the second LNS/LG domain from neurexin 1α: Ca²⁺ binding and the effects of alternative splicing

Neurexins mediate protein interactions at the synapse, playing an essential role in synaptic function. Extracellular domains of neurexins, and their fragments, bind a distinct profile of different proteins regulated by alternative splicing and Ca²⁺. The crystal structure of n1α_LNS#2 (the second LNS/LG domain of bovine neurexin 1α) reveals large structural differences compared with n1α_LNS#6 (or n1β_LNS), the only other LNS/LG domain for which a structure has been determined. The differences overlap the so-called hyper-variable surface, the putative protein interaction surface that is reshaped as a result of alternative splicing. A Ca²⁺-binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca²⁺-binding site in n1α_LNS#2 has low affinity (K_d∼400 μM). Ca²⁺ binding ceases to be measurable when an 8- or 15-residue splice insert is present at the splice site SS#2 indicating that alternative splicing can affect Ca²⁺-binding sites of neurexin LNS/LG domains. Our studies initiate a framework for the putative protein interaction sites of neurexin LNS/LG domains. This framework is essential to understand how incorporation of alternative splice inserts expands the information from a limited set of neurexin genes to produce a large array of synaptic adhesion molecules with potentially very different synaptic function.