Structures of neurexophilin–neurexin complexes reveal a regulatory mechanism of alternative splicing

Abstract

Neurexins are presynaptic, cell‐adhesion molecules that specify the functional properties of synapses via interactions with trans‐synaptic ligands. Neurexins are extensively alternatively spliced at six canonical sites that regulate multifarious ligand interactions, but the structural mechanisms underlying alternative splicing‐dependent neurexin regulation are largely unknown. Here, we determined high‐resolution structures of the complex of neurexophilin‐1 and the second laminin/neurexin/sex‐hormone‐binding globulin domain (LNS2) of neurexin‐1 and examined how alternative splicing at splice site #2 (SS2) regulates the complex. Our data reveal a unique, extensive, neurexophilin–neurexin binding interface that extends the jelly‐roll β‐sandwich of LNS2 of neurexin‐1 into neurexophilin‐1. The SS2A insert of LNS2 augments this interface, increasing the binding affinity of LNS2 for neurexophilin‐1. Taken together, our data reveal an unexpected architecture of neurexophilin–neurexin complexes that accounts for the modulation of binding by alternative splicing, which in turn regulates the competition of neurexophilin for neurexin binding with other ligands.

Alternative splicing controls teneurin-latrophilin interaction and synapse specificity by a shape-shifting mechanism

“…In addition to TEN2, numerous proteins such as DSCAMs, protocadherins, neurexins and neuroligins use alternative splicing for diversifying their functions, such as their ability to bind ligands [49][50][51][52] . In most proteins, the alternatively spliced sites localize to the ligandbinding site in order to directly enable or disturb ligand binding 42,43,53 . Thus, it is unusual that the LPHN3 binding site is localized away from the alternatively spliced sequence.…”

“…1d-f, 2c, d). This observation is very surprising because in other protein-ligand interactions regulated by alternative splicing, the alternatively spliced sequence is located at the ligand-binding interface 42,43 . Thus, in the case of the TEN2/LPHN3 interaction, alternative splicing regulates this interaction remotely in a manner that was previously not described.…”

Deorphanizing FAM19A proteins as pan-neurexin ligands with an unusual biosynthetic binding mechanism

“…Recombinant protein expression and purification for size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) For recombinant protein expression for SEC-MALS, the Bac-Mam system (Dukkipati et al, 2008;Goehring et al, 2014;Wilson et al, 2019) was used. Constructs were cloned into the pEG BacMam vector and transformed into MAX Efficiency DH10Bac Competent Escherichia coli (Invitrogen) to generate recombinant bacmid DNA.…”

Structure of the teneurin-latrophilin complex: Alternative splicing controls synapse specificity by a novel mechanism

“…In addition to Tenm2, numerous proteins such as DSCAMs, protocadherins, neurexins and neuroligins use alternative splicing for diversifying their functions, such as their ability to bind ligands (Aoto et al, 2013;Fuccillo et al, 2015;Irimia et al, 2014;Thalhammer et al, 2017). However, in most proteins, the alternatively spliced sites localize to the ligand-binding site in order to directly enable or disturb ligand binding (Arac et al, 2007;Shen et al, 2008;Wilson et al, 2019). Thus, it is unusual that the Lphn3 binding site is localized away from the alternatively spliced sequence.…”

“…A striking observation from the Tenm2/Lphn3 complex structure was that the Lphn3 binding site on Tenm2 is located distal to the alternatively spliced sequence in the Tenm2 β -propeller. This observation is very surprising because in other ligand interactions regulated by alternative splicing, the alternatively spliced sequence is usually located at the ligand-binding interface (Arac et al, 2007;Wilson et al, 2019). Thus, the Tenm2/Lphn3 interaction is the first case to our best knowledge in which alternative splicing regulates a ligand interaction remotely.…”