The reelin-N domain of F-spondin assumes a variant Ig-like fold in its core structure while its N-terminal variable region and long CD, D′D″ and FG loops form a unique additional layer. The conformational difference between the two reelin-N domains within an asymmetric unit is substantial and is closely associated with their extensive interaction through primarily their additional layers (). Across their asymmetric interface there are at least 15 pairwise interactions including seven salt bridges (). The residues contributing to the salt bridges, such as Arg91, Glu134, Arg139 and Arg167 are highly conserved within F-spondins (). There are also other highly specific and conserved hydrogen bonds such as those involving Asn121, Gln123, Thr151 and Gln165. The buried surface area between the two reelin-N domains is as high as 1781 A2
, well within the range of being physiologically significant 22
. Moreover, the interface's shape complementarity 23
, the measurement of geometric match between two juxtaposed surfaces, is as high as 0.65, a value within the range of antibody-antigen interface complementarities 23
. These data all imply that the observed asymmetric reelin-N domain dimer may represent a physiological entity, rather than a pure crystallographic packing artifact. However, the interface without extensive hydrophobic interactions is unlikely to hold the homophilic dimer in solution (). The monomeric reelin-N domain found in the size-exclusion chromatography could result from low affinity bonding and a fast off rate. Highly specific and low-affinity homophilic or heterophilic interactions are commonly found in adhesion molecules, which play important roles in molecular recognition and adhesion 24; 25; 26
. One similar and well characterized interface is the one between CD2 and CD58, which has extensive salt links and hydrogen bonds and only a small patch of hydrophobic interactions 27
. The heterophilic adhesion dimer of CD2/CD58 is well characterized with low affinity and very fast Koff
and Kon28; 29
Figure 5 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 (more ...)
Considering that its buried interface area and the interface's shape complementarity are both significantly larger than that of the CD2-CD58 heterodimer 27
, we propose that the asymmetric reelin-N domain dimeric assembly found in the structure () represents a weak dimer. The question is why the reelin-N domain can potentially form such a weak dimer. It is conceivable that such a dimer can bridge intercellular or cell-matrix interaction through two F-spondin molecules. Another possibility is that a dimer has the advantage of providing multivalency, which may increase binding strength and/or combine the binding activities of different domains 30
. The roles of oligomerization of thrombospondins and other ECM proteins have been studied extensively 30
. The importance of multivalence is also true for individual domains of these ECM proteins. Recently, we have shown that heparins can bind TSPN domains either individually or as dimers for more extensive interactions 31
Our experimental data () indicate that the reelin-N domain is capable of binding heparin with relatively high affinity. The reelin-N domain structure revealed in this study now allows us to map the possible binding sites on the domain for heparin. A cluster of positively-charged residues is regarded as a good indicator of a heparin-docking site, which involves mostly ionic interactions between heparin's sulfate groups and the protein's positively charged residues 32
. A highly conserved sequence motif (R138RRTR, see ) is located on the D″E loop and the beginning of the D″ strand, which are on the top of reelin-N domain (). This sequence motif creates the single largest positively charged patch on the molecular surface of the reelin-N domain and probably functions as the primary heparin-binding site (). Other residues that are likely to be engaged are Lys166 and Arg167, which are on the FG0 (or FG0′) loop, and also located on the top of the reelin-N domain ( and ). Despite the fact that the FG loop changes its conformation dramatically from one molecule to the other (), Lys166 and Arg167 are always positioned near the proposed primary heparin-binding site, and therefore should enhance heparin binding. In molecule A, these two residues, together with Arg46, form the second positively charged patch on the protein's surface ( and ). The distance between this site and the primary binding site is about 16 Å, a distance that corresponds to about one turn of the heparin right-handed helix. The two binding sites with such a separation help to bind heparin collectively on one side of the helical molecule. In molecule B, Lys166 and Arg167 move closer to the primary binding site, forming an extended primary positively-charged patch ( and ). Therefore, Lys166 and Arg167 seem to function in coordination with the prominent positively-charged sequence motif (R138RRTR), irrespective of the conformational variation.
Interestingly, the potential asymmetric reelin-N dimer seems to bring the heparin-binding sites from two individual monomers together to create a groove that is fully lined with positively charged residues, with a second extended positively charged patch on the other side of the dimer (). On one hand, an extensive heparin-binding site can certainly increase heparin-binding affinity. On the other hand, a heparin sitting along one of these extended binding sites could externally bridge two reelin-N domains and help to stabilize the asymmetric dimer. The scenario seems to be reminiscent of what has been observed in the two dimerization modes induced by heparin binding of the N-terminal domain of thrombospondin-1 31
. However, the potential role of the weak asymmetric reelin-N domain dimer formation in binding to heparin, and possible other ligands, remains to be explored and verified.
The functions of the GAG-binding site of F-spondin or its reelin-N domain have not been fully established. The GAG binding activity of the reelin-N domain of F-spondin may serve to anchor F-spondin to ECM in such a way that the spondin domain and the TSRs are available for interactions with cells or possibly visa verse
. The orientation of the TSRs in such a scenario could be important for the appropriate guidance of the growth cones of commissural axons. This orientation may also facilitate the proteolytic processing of F-spondin that releases the TSRs from the intact molecule 2
. After proteolysis, the remaining fragment, designated the reespo domain, would contain the reelin-N domain and the spondin domain 4
. The reespo domain reportedly promotes the outgrowth of sensory neurons 4
. The reelin-N and spondin domains of F-spondin also mediate binding to APP 8; 9
. Like the reelin-N domain of F-spondin, APP binds to GAGs 33
. These data raise the possibility that the GAG binding activity of the reelin-N domain of F-spondin may facilitate its interaction with APP by co-localizing the two proteins on the cell surface.