Crystal Structure of the Ligand Binding Domain of Netrin G2

Abstract

Netrin G proteins represent a small family of synaptic cell adhesion molecules related to netrins and to the polymerization domains of laminins. Two netrin G proteins are encoded in vertebrate genomes, netrins G1and G2, which are known to bind the leucine-rich repeat proteins netrin G ligands (NGLs) 1 and 2, respectively. Netrin G proteins share a common multi-domain architecture comprising an laminin N-terminal (LN) domain followed by three Laminin-EGF (LE) domains and a C′ region containing a GPI anchor. Here we use deletion analysis to show that the LN domain region of netrin Gs contains the binding site for NGLs to which they bind with 1:1 stoichiometry and sub-micromolar affinity. Netrin Gs are alternatively spliced in their LE domain regions, but the binding region, the LN domain, is identical in all splice forms. We determined the crystal structure for a fragment comprising the LN domain and domain LE1 of Netrin G2 by Sulfur-SAD phasing and refined it to 1.8Å resolution. The structure reveals an overall architecture similar to laminin α chain LN domains, but includes significant differences including a Ca2+ binding site in the LN domain. These results reveal the minimal binding unit for interaction of netrin Gs with NGLs, define structural features specific to netrin Gs, and suggest that netrin G alternative splicing is not involved in NGL recognition.

Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data

“…Details of the production of proteins and crystals for the HK9 S , netrin G2, CysZ and TorT-TorS S analyses are described in the supplementary material of our previous paper (Liu et al, 2012) and in the associated structure reports (Brasch et al, 2011;Moore & Hendrickson, 2012;Qi et al, 2013). Many of the procedures used for data collection, data reduction and structural analysis have also been described previously.…”

“…9. Structures from three of these analyses, and their associated biological implications, are described in full elsewhere (Brasch et al, 2011;Moore & Hendrickson, 2012;Qi et al, 2013) and descriptions for the other two are in preparation; the initial four structures were also shown in our previous account of this work (Liu et al, 2012). The five structures range from 127 to 1200 ordered residues, are in crystal lattices from monoclinic to trigonal to tetragonal, have resolution limits from medium at 2.3 Å to low at 2.8 Å and contain systems such as a molecular machine (DnaK), a membrane protein (CysZ) and a protein-protein complex (TorT-TorS s ).…”

Building branched tissue structures: from single cell guidance to coordinated construction

“…Vertebrate netrin protein members include secreted proteins netrin 1-4 [39] and glycosyl phosphatidylinositol (GPI)-anchored netrinG1 and G2 [40,41]. Here, we focus on netrins1-3, as netrin-4 and netrinG proteins are not diffusible guidance cues.…”

Control of neural circuit formation by leucine-rich repeat proteins

“…Netrin-G1 binding to LAR is modulated by alternative splicing of Netrin-G1 [43], which can generate at least 10 isoforms [44–46]. Alternative splicing of Netrin-G2 does not affect binding to NGL-2 [47], suggesting that Netrin-G splicing serves to regulate cis interactions. Thus, like LRRTMs, different NGL family members interact with transmembrane or GPI-anchored presynaptic partners, and as suggested for the LRRTM4-glypican interaction, the recruitment of additional cis co-receptors is required for Netrin-G-mediated presynaptic differentiation.…”