Structzal analysis of receptor-ligand interactions in cell signaling

在脑发育过程中，一个大的细胞外糖蛋白reelin指导神经元的迁移，并在层的形成中起基础作用。它是由8个串联重复的~380个残基的单位，称为reelin重复，它有一个中央EGF模块两侧的两个同源的亚重复序列没有明显的序列相似性，以已知的蛋白质。小鼠reelin重复序列3的1.9A晶体结构揭示了亚重复序列呈现与碳水化合物结合结构域具有意想不到的结构相似性的β-卷曲折叠。尽管有EGF模块的干预，两个子域直接接触，导致紧凑的整体结构。电子显微镜下，包含第3至第6个重复序列的四域片段能够诱导神经元中Dab 1磷酸化，其呈杆状，具有四个斑点。此外，通过单粒子断层扫描获得的片段的3D分子包膜可以与四个串联的重复3原子结构相匹配，从而首次瞥见了这类重要的信号分子的结构单元。我们接下来发现，受体结合和随后的Dab 1磷酸化仅发生在跨越第五和第六个reelin重复序列（R5-6）的片段中。单体片段表现出次优水平的信号传导活性，人工寡聚化导致活性增加10倍，表明高阶多聚化在生理性缫丝素中的至关重要性。来自R5-6片段的2.0A晶体结构不仅揭示了其中两个重复序列以相同取向并排排列的独特结构域排列，而且还揭示了结合的Zn离子的意外存在。R5-6的结构引导的丙氨酸突变显示，两个赖氨酸残基（Lys 2360和Lys 2467）构成了受体中的LDLR A类模块的中心结合位点，表明与内吞脂蛋白受体之间共享的配体识别模式具有很强的相似性。

A large extracellular glycoprotein reelin directs neuronal migration during the brain development and play fundamental role in the layer formation. It is composed of eight tandem repeats of ~380-residue unit, termed reelin repeat, which has a central EGF module flanked by two homologous subrepeats with no obvious sequence similarity to known proteins. The 1.9A crystal structure of mouse reelin repeat 3 reveals that the subrepeat assumes a β-jelly-roll fold with unexpected structural similarity to carbohydrate binding domains. Despite the intervention by an EGF module, two subdomains make direct contact resulting in a compact overall structure. Electron micrographs of four-domain fragment encompassing 3rd to 6th repeats, which is capable of inducing Dab1 phosphorylation in neuron, show rod-like shape with four blobs. Furthermore, 3D molecular envelope of the fragment obtained by single-particle tomography can be fitted with four concatenated repeat 3 atomic structures, giving the first glimpse of the structural unit for this important class of signaling molecule.We next found that both receptor binding and subsequent Dab1 phosphorylation occur solely in the segment spanning the fifth and sixth reelin repeats (R5-6). Monomeric fragment exhibited a suboptimal level of signaling activity and artificial oligomerization resulted in a 10-fold increase in activity, indicating the critical importance of higher-order multimerization in physiological reelin. A 2.0A crystal structure from the R5-6 fragment revealed not only a unique domain arrangement wherein two repeats were aligned side by side with the same orientation, but also the unexpected presence of bound Zn ions. Structure-guided alanine mutagenesis of R5-6 revealed that two Lys residues (Lys2360 and Lys2467) constitute a central binding site for the LDLR class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.