The Structure of the Reelin-N Domain. The stereo views of ribbon drawings of monomer A (A) and B (B) on the left side and their topology schemes on the right side. In the ribbon drawings the β strands in the front β sheet are colored in green while those in the back β sheet are colored in cyan. The secondary structures that form additional layer (see text) and represent dramatic conformational change between the two monomers are colored in dark yellow. A segment on the CD loop of monomer A that forms two hydrogen bonds to the A0 strand is drawn in thick dark yellow line. The four cysteine residues that form two disulfide bonds as well as some of positively charged residues that may involve in heparin-binding are drawing in ball-and-stick format. Figure 2 and 4 were prepared using the program MOLSCRIPT 38.

The Asymmetric Reelin-N Domain Dimer and its Interface. (A) A ribbon diagram drawing of the asymmetric reelin-N domain dimer in a crystallographic asymmetric unit. The color codes for the secondary structures of the two monomers and individual residues are the same as those in Figure 2. The disulfide bond forming cysteine residues and some of the potential heparin-binding residues shown in Figure 2 are also drawing in ball-and-stick format in this figure for comparing molecules' orientations in the two figures. (B) A stereo view of the interface of reelin-N domain dimer. The residues involved in the high-specific interaction are drawn in the ball-and-stick format. The interactions across the interface are very asymmetric for the homodimer.

The Reelin-N Domain Binds Heparin. (A) A schematic diagram of the domain structure of F-spondin showing the relative position of the reelin-N domain, the spondin domain (FS1+2), which includes two sequential repeats designated fs1 and fs2, and the six TSRs. (B) Size-exclusion chromatography of the recombinant reelin-N domain. The recombinant protein was first purified using Ni-affinity chromatography. Six protein standards were used to calibrate the column in a separated run (data not shown). The molecular mass of the protein from the principal peak was calculated to be 21.0 kDa based on the calibration. (C) SDS PAGE of the reelin-N domain after Ni-affinity chromatography (left lane) and heparin-Sepharose affinity chromatography. The initial buffer included 100 mM NaCl and 20 mM HEPES at pH 7.8. Whereas the majority of the reelin-N domain was eluted from the heparin-Sepharose column in buffer containing 450 mM NaCl, some protein was also present in the 550 mM NaCl eluate.

Multiple Sequence Alignment of Reelin-N Domains across Different Species. Based on the reelin-N domain structures of the two monomers, the secondary structures are indicated with arrows (β strands) and rods (α helices) below the appropriate sequences, respectively. Identical residues are highlighted in red and highly similar residues in brown. The sequences used in the alignment include human F-spondin (gi:110347423), rat F-spondin (gi:544353), chicken F-spondin (gi:45382337), Xenopus F-spondin (gi:544354), zebrafish F-spondin-1 (gi:18859413), zebrafish F-spondin-2 (gi:2529227), amphiF-spondin (gi:3319874), Drosophila melanogaster F-spondin (gi: 19922086), human reelin (gi:20139420), rat reelin (gi:149046582) and C.elegans hypothetical protein F10E7.4 (gi:32564253). The first 14 N-termnal residues (FSDETLDKVPKSEG) and the last 10 C-terminal residues (DSTFDGVTDK) in the expression construct of the human F-spondin reelin-N domain in this study were not used for the alignment. Most of these residues are disordered in the structure. The two highly conserved disulfide bonds are marked in lines connecting paired cysteines. For structural determination, the site of mutation of L82/M is marked with “ * ”.

The Surface Potentials and Mapping of Heparin-binding Sites. (A) The electrostatic potential surface representation of the two individual reelin-N domains, monomer A (left) and monomer B (right). Some positively charged residues that may form heparin-binding sites are mapped to their surface to show their relative orientation to those in Figure 2. (B) The electrostatic potential surface representation of the reelin-N domain dimer. The two orientations of the dimer is related by a 160 degrees of rotation along the horizontal axis. The first one is similar to that shown in Figure 4A. Some potential heparin-binding residues from two monomers are mapped to the dimer surface. Figure 5 was prepared with the program GRASP 39.