Structzal analysis of receptor-ligand interactions in cell signaling

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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.