Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY 10032, USA.
J Mol Biol. 2011 Dec 16;414(5):723-34. doi: 10.1016/j.jmb.2011.10.030. Epub 2011 Oct 25.
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 G1 and G2, which are known to bind the leucine-rich repeat proteins netrin G ligand (NGL)-1 and NGL-2, respectively. Netrin G proteins share a common multi-domain architecture comprising a laminin N-terminal (LN) domain followed by three laminin epidermal growth factor-like (LE) domains and a C' region containing a glycosylphosphatidylinositol 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 single-wavelength anomalous diffraction phasing and refined it to 1.8 Å resolution. The structure reveals an overall architecture similar to that of laminin α chain LN domains but includes significant differences including a Ca(2+) 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.
神经导向因子 G 蛋白是一小类突触细胞黏附分子，与神经导向因子和层粘连蛋白的聚合结构域有关。脊椎动物基因组中编码了两种神经导向因子 G 蛋白，即神经导向因子 G1 和 G2，它们分别与富含亮氨酸重复的蛋白神经导向因子 G 配体（NGL）-1 和 NGL-2 结合。神经导向因子 G 蛋白具有共同的多结构域架构，包括层粘连蛋白 N 端（LN）结构域，其后是三个层粘连蛋白表皮生长因子样（LE）结构域和一个含有糖基磷脂酰肌醇锚的 C'区。在这里，我们通过缺失分析表明，神经导向因子 G 的 LN 结构域区域包含与 NGL 结合的结合位点，它们以 1:1 的化学计量和亚微米亲和力结合。神经导向因子 G 在其 LE 结构域区域发生选择性剪接，但结合区域，即 LN 结构域，在所有剪接形式中都是相同的。我们通过硫单波长异常衍射相分析确定了神经导向因子 G2 的 LN 结构域和 LE1 结构域的一个片段的晶体结构，并将其精修至 1.8 Å分辨率。该结构揭示了一种与层粘连蛋白α链 LN 结构域相似的整体结构，但包括显著差异，包括 LN 结构域中的 Ca(2+)结合位点。这些结果揭示了神经导向因子 G 与 NGL 相互作用的最小结合单元，定义了神经导向因子 G 的结构特征，并表明神经导向因子 G 的选择性剪接不参与 NGL 的识别。
