Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors

doi:10.1073/pnas.0700438104

Structure of a receptor-binding fragment of reelin and mutational analysis reveal a recognition mechanism similar to endocytic receptors

*Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan; and
^†Department of Biomedical Science, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabedori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan

Edited by Thomas C. Südhof, University of Texas Southwestern Medical Center, Dallas, TX, and approved May 6, 2007 (received for review January 17, 2007)

Abstract

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-Å 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.

Footnotes

^‡To whom correspondence should be addressed. E-mail: takagi{at}protein.osaka-u.ac.jp
Author contributions: N.Y. and J.T. designed research; N.Y., T.N., T.K., Y.N., and M.H. performed research; N.Y., T.N., M.H., and J.T. analyzed data; N.Y., T.N., M.H., and J.T. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates and structural factors have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2E26).
This article contains supporting information online at www.pnas.org/cgi/content/full/0700438104/DC1.
Abbreviations:

LDLR,

low-density lipoprotein receptor;

ApoE,

apolipoprotein E;

ApoER2,

ApoE receptor 2;

Dab1,

disabled-1;

LA,

LDLR class A;

VLDLR,

very LDLR.