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. 2007 Jun 12;104(24):9988-93.
doi: 10.1073/pnas.0700438104. Epub 2007 Jun 4.

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

Norihisa Yasui  1 , Terukazu Nogi, Tomoe Kitao, Yoshimi Nakano, Mitsuharu Hattori, Junichi Takagi
Affiliations

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

Norihisa Yasui et al. Proc Natl Acad Sci U S A. .

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-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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Determination of minimum regions essential for reelin–receptor interaction. (A) A series of deletion constructs (shown schematically at the top) of reelin containing a C-terminal tag (black rectangle) were expressed in 293T cells and subjected to a solid-phase binding assay, using hGH-ApoER2 fusion protein. Culture supernatants were incubated with receptor-immobilized (lane R) or unmodified (lane C) beads and bound proteins were analyzed by Western blot analysis, using anti-Myc antibody. Ni-NTA agarose pull-down (lane Ni) was used to estimate the amounts of total recombinant fragments present in the medium. (B) LA module fragments of ApoER2 extracellular domain were expressed as hGH fusion protein, captured on anti-hGH antibody beads, and incubated with culture supernatants containing the R5–6 fragment. Bound R5–6 fragments were detected by anti-Myc antibody (Upper). The same membranes were reprobed with anti-hGH antisera to compare the expression levels of the different constructs (Lower).
Fig. 2.
Fig. 2.
Dab1 phosphorylation activity. (A) Mouse cortical neurons were incubated for 20 min with the purified R5–6 fragment at the indicated concentration or with culture supernatant from the cells transfected with (WT) or without (mock) full-length reelin. Phosphorylated and total Dab1 were detected by Western blot analysis, using 4G10 and anti-Dab1, respectively. (B) The biotinylated R5–6 fragment was oligomerized by streptavidin (see SI Fig. 11) and tested for Dab1 phosphorylation activity, using cultured neurons. The concentrations of R5–6 fragments are shown in nM with respect to monomers, using a molecular mass value of 85,000; the concentrations of WT reelin are expressed as the dilution fold of the culture supernatant.
Fig. 3.
Fig. 3.
Crystal structure of R5–6. (A) Overall structure of R5–6 shown in a ribbon model, and viewed from two different angles. Subdomains in each reelin repeat are differently colored: subrepeat A (cyan), EGF (green), and subrepeat B (magenta). Ca and Zn ions are shown as gold and purple spheres, respectively. Disulfide bonds (yellow) and four N-glycan chains (gray) are shown in stick models. All structural images were prepared with PyMOL (http://pymol.sourceforge.net). (B) Zn-binding sites. Close-up view of site 1 (Left) and site 2 (Right) are shown. Both Zn2+ (purple spheres) were coordinated by His and Glu side chains of an R5–6 molecule (cyan). In site 1, a Glu side chain from the symmetry mate molecule (gray) completed the coordination, whereas in site 2, two water molecules (red spheres) were bound.
Fig. 4.
Fig. 4.
Mapping the amino acid residues critical for receptor binding. (A) The binding of R5–6 mutants to hGH-ApoER2 EC was analyzed by a solid-phase binding assay. Receptor-bound (Upper) and Ni-captured (Lower) fragments were detected by Western blot analysis, using anti-Myc antibody. (B) Full-length reelin samples, with the indicated mutations, were tested for receptor-binding activity (Upper). CR-50 pull down confirmed that roughly equal amounts of reelin were present in the culture supernatants (Lower). (C) Dab1 phosphorylation activities of the full-length reelin mutants. (D) Lys residues mutated in this study and the single free cysteine residue are shown in blue and yellow stick models, respectively, on the Cα backbone of R5–6.
See this image and copyright information in PMC

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