Regulation of Neurexin 1β Tertiary Structure and Ligand Binding through Alternative Splicing

Abstract

Neurexins and neuroligins play an essential role in synapse function, and their alterations are linked to autistic spectrum disorder. Interactions between neurexins and neuroligins regulate inhibitory and excitatory synaptogenesis in vitro through a "splice-insert signaling code." In particular, neurexin 1beta carrying an alternative splice insert at site SS#4 interacts with neuroligin 2 (found predominantly at inhibitory synapses) but much less so with other neuroligins (those carrying an insert at site B and prevalent at excitatory synapses). The structure of neurexin 1beta+SS#4 reveals dramatic rearrangements to the "hypervariable surface," the binding site for neuroligins. The splice insert protrudes as a long helix into space, triggers conversion of loop beta10-beta11 into a helix rearranging the binding site for neuroligins, and rearranges the Ca(2+)-binding site required for ligand binding, increasing its affinity. Our structures reveal the mechanism by which neurexin 1beta isoforms acquire neuroligin splice isoform selectivity.

Mutational analysis of the neurexin/neuroligin complex reveals essential and regulatory components

“…S1). Ca 2ϩ binding in Nrxn (by D137, V154, I236 and N238 for ␤-Nrxn), agrin, and laminins represents a rigid ''socket'' with no f lexible states as in EF hands (23), consistent with unchanged CD spectra of Nrxn and agrin (13,38) and crystal data (15)(16)(17)39). This rigidity allowed us to transfer epitopes from other LNS domains to Nrxn: ␤N238G mimics the Ca 2ϩ coordination of Nrxn LNS2 but reduced Ca 2ϩ affinity of the Nrxn/Nlgn complex Ϸ200-fold (Fig.…”

“…Because the complex still forms with this mutation at physiological [Ca 2ϩ ], it appears unlikely that calcium affinity itself defines splice-code-specific pairings (21). Rather, crystal and NMR data revealed that insert SS4 exists in an equilibrium of two conformations (39,40). The inactive form is stabilized by coordinating Ca 2ϩ with high affinity (39), whereas binding follows after unfolding of SS4 (40).…”

“…Rather, crystal and NMR data revealed that insert SS4 exists in an equilibrium of two conformations (39,40). The inactive form is stabilized by coordinating Ca 2ϩ with high affinity (39), whereas binding follows after unfolding of SS4 (40). Therefore, the 50% reduction in complex formation when SS4 is present could sufficiently be explained by a reduced availability of the active form.…”

Characterization of the neuroligin gene family expression and evolution in zebrafish

“…They bind to presynaptic alpha and beta forms of Neurexins (Ichtchenko et al, 1995(Ichtchenko et al, , 1996Missler et al, 1998;Boucard et al, 2005) and the alternative splicing of both protein families controls their binding affinities (Boucard et al, 2005;Chih et al, 2006;Comoletti et al, 2006;Fabrichny et al, 2007;Koehnke et al, 2008a,b;Shen et al, 2008). The current data suggest that Neurexin-Neuroligin binding is governed by a complex code that is based on the type of isoforms and splice variants involved, calcium binding, and glycosylation (Sudhof, 2008).…”

Calsyntenin-3 Molecular Architecture and Interaction with Neurexin 1α

“…Purified hexahistidinetagged bovine neurexin 1␣ and its fragments as well as hexahistidine-tagged rat neuroligin 2 (NL2) containing insert ϩA2 were produced in a manner similar to that described previously (16). Recombinant rat neurexin 1␤ was produced as described (26). For analytical size exclusion chromatography, proteins were loaded on a Superdex 200 PC 3.2/30 column in 25 mM Tris, pH 8.0, 120 mM NaCl, 5 mM CaCl 2 and run at 0.08 ml/min.…”