Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors.
Reelin, a large secreted protein implicated in the cortical development of the mammalian brain, is composed of eight tandem concatenations of "reelin repeats" and binds to neuronal receptors belonging to the low-density lipoprotein receptor gene family. We 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 2.0-A 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 (Lys-2360 and Lys-2467) constitute a central binding site for the low-density lipoprotein receptor class A module in the receptor, indicating a strong similarity to the ligand recognition mode shared among the endocytic lipoprotein receptors.
Yasui
Norihisa
N
Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Nogi
Terukazu
T
Kitao
Tomoe
T
Nakano
Yoshimi
Y
Hattori
Mitsuharu
M
Takagi
Junichi
J
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D015816:Cell Adhesion Molecules, Neuronal / Q000737:chemistry*; D002460:Cell Line; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D002999:Clone Cells; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D018274:Electroporation; D016326:Extracellular Matrix Proteins / Q000737:chemistry*; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006801:Humans; D026502:LDL-Receptor Related Proteins; D008024:Ligands; D051379:Mice; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D018110:Receptors, Lipoprotein / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D000091042:Reelin Protein; D012697:Serine Endopeptidases / Q000737:chemistry*; D015032:Zinc / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D015816:Cell Adhesion Molecules, Neuronal; C115785:DAB1 protein, human; D004220:Disulfides; D016326:Extracellular Matrix Proteins; D026502:LDL-Receptor Related Proteins; D008024:Ligands; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D018110:Receptors, Lipoprotein; D011993:Recombinant Fusion Proteins; D000091042:Reelin Protein; C098773:low density lipoprotein receptor-related protein 8; D018698:Glutamic Acid; D006639:Histidine; C000717127:RELN protein, human; C000717128:Reln protein, mouse; D012697:Serine Endopeptidases; D015032:Zinc