Crystal Structure of the Second LNS/LG Domain from Neurexin 1α

Abstract

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 Ca 2؉ . The crystal structure of n1␣_LNS#2 (the second LNS/LG domain of bovine neurexin 1␣) reveals large structural differences compared with n1␣_LNS#6 (or n1␤_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 Ca 2؉ -binding site is revealed at the center of the hyper-variable surface next to splice insertion sites. Isothermal titration calorimetry indicates that the Ca 2؉ -binding site in n1␣_LNS#2 has low affinity (K d ϳ 400 M). Ca 2؉ 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 Ca 2؉ -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.Neurexins are multidomain cell surface proteins found in the brain at the synapse (recently reviewed in Refs. 1-4). In mammals, the neurexin family consists of three genes that each encode an ␣-and a ␤-neurexin (5-8). Protruding into the synaptic cleft, the extracellular domain of ␣-neurexins consists of six LNS/LG 2 domains, whereas ␤-neurexins contain only a single LNS/LG domain preceded by a short ␤-neurexin-specific sequence (nomenclature and abbreviations defined below and in Fig. 1). There is increased similarity between the first, third, and fifth LNS/LG domains as well as between the second, fourth, and sixth LNS/LG domains. Nevertheless the sequence identity is low between neurexin LNS/LG domains (20 -25%).Neurexins facilitate synapse functioning and neurotransmitter release through protein-protein interactions (9 -18). Neurexins interact inside the neuron with the cytomatrix of the active zone and outside the neuron with proteins at the synaptic cleft. Single LNS/LG domains of neurexins are sufficient to bind ligands (though not necessarily optimal), binding is Ca 2ϩ -dependent, and individual LNS/LG domains display distinct ligand-binding specificities (14, 17, 19 -22). The second LNS/LG domain of neurexin 1␣, neurexin 1␣_LNS#2 (n1␣_LNS#2), interacts with the extracellular matrix protein ␣-dystroglycan (22) and neurexophilin, a small neuropeptidelike protein (20,23). LNS#6 in neurexin 1␣ (n1␣_LNS#6) and the identical LNS domain in neurexin 1␤ (n1␤_LNS) interact with neuroligins, a family of post-synaptic cell surface proteins (9,14,17,24,25), ␣-latrotoxin, a spider neurotoxin triggering massiv...

Crystal Structure of the N-terminal NC4 Domain of Collagen IX, a Zinc Binding Member of the Laminin-Neurexin-Sex Hormone Binding Globulin (LNS) Domain Family

“…6, B and C), and neurexin 1␣_LNS#2 domains reveals not only the overall homology but also common features in the metal binding architecture. The striking similarity is that the same connecting loops are involved in metal ion binding and that these metal ions, except for the Zn 2ϩ in NC4, have been shown to affect ligand binding (23,51,(53)(54)(55).…”

“…The Ca 2ϩ ion in the Gas6 G domain pair interface (25) as well as the one in the calnexin convex sheet (24) is structural rather than functional. On the other hand, the Ca 2ϩ ions in the laminin LG5, the agrin G3, and the neurexin 1␣_LNS#2 domains are strictly required for ␣-dystroglycan binding (20,23,26,53). In SHBG, a Ca 2ϩ in a position similar to that in Gas6 and a Zn 2ϩ affecting estradiol binding have been identified (21,51).…”

“…However, the NC4 Zn 2ϩ data in this study and the recent agrin G3 (26) and neurexin 1␣_LNS#2 (23) Ca 2ϩ binding data together confirm that low affinity metal ions are commonly found on the LNS domain ␤-sandwich edges, the common ligand interaction regions. These ions can be crucial for ligand binding despite the low affinity of their interaction per se (23,26). Furthermore incomplete coordination of the metal has been reported also in other LNS domains (20,50,51).…”

“…In a very recent report, the second LNS/LG domain of neurexin 1␣ (1␣_LNS#2; Ref. 23) was shown to have a Ca 2ϩ ion in a similar position and with analogous coordination to laminin LG5 and agrin G3 domains. However, the neurexin 1␣_LNS#2 has only three protein ligands due to the lack of counterpart for the aspartate in the ␤12-␤13 loop.…”

“…On the basis of amino acid sequence conservation and the apparent similarities of the secondary structural elements, it was predicted that the TSPN module is homologous with pentraxins and with members of the lamininneurexin-sex hormone binding globulin (LNS) domain family (18,19). Three-dimensional structures of LNS modules from the laminin ␣2 chain (␣2LG5) (20), sex hormone binding globulin (SHBG) (21), neurexins 1␤ (22) and 1␣ (23), calnexin (24), growth arrest-specific protein Gas6 (25), agrin (26), and the N-terminal domain of thrombospondin-1 (TSPN-1) (27) along with structures of the pentraxins serum amyloid P component (28) and C-reactive protein (29,30) are now available. All members of the superfamily share a ␤-sandwich fold with a convex and a concave sheet, each comprised of six or seven antiparallel ␤-strands.…”