Structure of Reelin repeat 8 and the adjacent C-terminal region

Abstract

Neuronal development and function are dependent in part on the several roles of the secreted glycoprotein Reelin. Endogenous proteases process this 400 kDa, modular protein, yielding N-terminal, central, and C-terminal fragments that each have distinct roles in Reelin's function and regulation. The C-terminal fragment comprises Reelin repeat (RR) domains seven and eight, as well as a basic stretch of 32 amino acid residues termed the C-terminal region (CTR), influences Reelin signaling intensity, and has been reported to bind to Neuropilin-1, which serves as a co-receptor in the canonical Reelin signaling pathway. Here, we present a crystal structure of RR8 at 3.0 Å resolution. Analytical ultracentrifugation and small-angle x-ray scattering confirmed that RR8 is monomeric and enabled us to identify the CTR as a flexible, yet compact subdomain. We conducted structurally informed protein engineering to design a chimeric RR8 construct guided by the structural similarities with RR6. Experimental results support a mode of Reelin-receptor interaction reliant on the multiple interfaces coordinating the binding event. Structurally, RR8 resembles other individual RRs, but its structure does show discrete differences that may account for Reelin receptor specificity toward RR6.

Figure 1

Reelin repeats share high sequence identity. (A) Top: schematic of full-length Reelin with three major cleavage products outlined. NT, N-terminal fragment; CF, central fragment; CT, C-terminal fragment. PDB and EMDB IDs are shown for regions for which structural information is available; domains are shaded gray if a high-resolution model is available. Bottom: schematic of RR8 construct used in this study. Subrepeat A is preceded by an N-terminal FLAG tag. Subrepeats A and B (light blue) both share a bacterial neuraminidase repeat (BNR) or Asp-box that forms a β-hairpin. An EGF-like domain separates subrepeats A and B. The C-terminal region (CTR) follows subrepeat B; it includes a Furin recognition site (dashed line) and ends at residue 3461. Human Fc is cloned in frame with the protein but is endogenously cleaved by Furin during expression of the WT protein. (B) Heatmap showing the degree of amino acid sequence identity between RRs. The repeats share ∼25–40% amino acid sequence identities. (C) Previously solved crystal structures of RR1, RR3, RR5, and RR6 show high similarity in tertiary structure when superimposed; RMSD values range from 1.3 to 2.8 Å.

Figure 2

Purification and crystal structure of RR8. (A) RR8 elutes as a single, symmetric peak corresponding to ∼50 kDa when compared with globular gel filtration standards on a Superdex 200 10/300 GL column. (B) SDS-PAGE followed by Coomassie staining in reducing conditions further shows that RR8 runs as a single band at 50 kDa. (C) The asymmetric unit of the solved crystal structure is composed of two chains, A (green) and B (cyan), and each chain comprises two subrepeats (brackets). The two chains exhibit noncrystallographic symmetry of 180° about a rotational axis (asterisk). Four N-linked glycans are represented as sticks, located on the back of model from the current perspective. Bottom left: the C-terminus of the crystal structure ends at residue S3430. Residues L3428 and V3429 were truncated at Cβ due to ill-defined electron density. Right: two Ca²⁺ ions are modeled on each chain (shown for chain B) as allowed by obvious electron density and coordination by electrostatically favorable interactions.

Figure 3

RR8 is a monomer in solution. (A) Sphere model of RR8 in the asymmetric unit, highlighting the crystallographic interface (red) between chains A (green) and B (blue). Yellow asterisks note the two N-linked glycosylation sites on each chain (N3185 and N3412). (B) Sedimentation velocity experiments conducted at 0.1, 0.3, and 0.9 OD (corresponding to 0.063, 0.19, and 0.56 mg/mL, respectively) show that RR8 sediments as a single species of 3.6 S with no evidence of oligomerization. (C) Of the signal measured during the sedimentation velocity analysis of RR8 at 0.3 OD, 99.3% is derived from molecular species of 49.6 and 52.6 kDa, both with an f/f0 of 1.43. (D) The crystal structure of RR8 modeled in its monomeric in-solution assembly. The monomer is elongated with its dimensions roughly being 60 × 35 × 30 Å, adopting a cylindrical shape. Subrepeat A is represented in beige; EGF-like domain is in pink; subrepeat B is in aquamarine; artifactually interfacing residues are in red.

Figure 4

Reelin’s CTR forms a conspicuous domain adjacent to RR8. (A) Superimposition of SAXS Damfilt bead model (spheres) and crystal structure of RR8 (cyan) with modeled CTR (orange). Note the excellent fit of the overlay. (B) Ab initio bead model fits the experimental data with a χ² = 1.16 (n = 368). (C) Crysol-calculated fit for the RR8 model with the appended CTR (+CTR) has a χ² = 1.12 (n = 567). (D) Crysol-calculated fit for the RR8 model without the CTR (ΔCTR) has a χ² = 1.36 (n = 567). Assigning a significance value of α = 1%, the Dammif bead model and RR8+CTR model fit the data with statistical significance, while the RR8ΔCTR model does not. Note the regions of data points that are consecutively positive (i) or negative (ii) to the fit in farthest right section of (D), compared with (B) and (C). The correlation map test (CorMap) values are also shown, representing the largest number of consecutive data points that are either positive or negative to the fit.

Figure 5

Comparison of RR6 and RR8 reveals global similarities but distinct differences. (A) RR6 (magenta) and RR8 (cyan) align with an RMSD of 1.63 Å. Box: outline of homologous residues K2467 and K3218, which are differently positioned and further reported in (C) and (D). K2467’s side chain is radially oriented and solvent exposed, while K3218’s side chain is buried into a nearby pocket. (B) Sequence comparison and conservation of the homologous loops reveals a highly conserved hydrophobic patch on RR6 that is not present on RR8. Nonpolar residues are bolded; basic residues are represented in blue; acidic residues are in red; polar, uncharged residues are in normal font. (C) Comparison of the loops interest. Several residues of RR8 are truncated at Cβ. (D) Superimposition of RR6 and RR8 shows that F2465 of RR6 occupies the pocket in which RR8’s K3218 is situated. (E) The two loops of interest are superimposed and shown in relation to ApoER2. RR6 clearly contacts the calcium coordination site on ApoER2 via K2467. The homologous lysine (K3218) on RR8 is pointed inward, away from any potential interaction with ApoER2. The dashed line highlights K3218’s 14.1 Å displacement when compared with K2467, and the arrows signify the approximate boundaries used in the design of the chimeric protein constructs (Fig. 6). The side chains for amino acid residues immediately outside of the boundaries are made visible; they align in near identical rotamers, suggesting the compatibility for a functional replacement of one loop for the other.

Figure 6

RR8 chimeras bind to neither ApoER2 nor VLDLR. (A) Amino acid sequence in loop of interest for RR6 and RR8, and the two loop swap constructs, RR8 QQ and RR8 HT. (B and C) RR8 QQ and RR8 HT elute as single peaks corresponding to ∼50 kDa when compared with globular gel filtration standards on a Superdex 200 10/300 GL column. SDS-PAGE followed by Coomassie staining in reducing conditions further shows that both RR8 QQ and RR8 HT run as single bands at 50 kDa. (D) ELISA-based binding assay between purified Reelin constructs coating the plate and either ApoER2-Fc at 10 nM (top) or VLDLR-Fc at 30 nM (bottom). Both experiments were conducted in technical triplicate, and RR8 WT functioned as a negative control, while Reelin’s central fragment (CF) served as a positive control. (E) Quantification of the ELISAs shows that only the positive control, Reelin CF, elicited a significant increase when compared with RR8 WT for both ApoER2-Fc (top) and VLDLR-Fc (bottom). In either experiment, no significant difference was observed for RR8 QQ and RR8 HT when compared with RR8 WT. ^∗∗p ≤ 0.01.