Fig 2: RMSF results for BEB3-BEB6 from MD simulations. Fluctuations include: (a) BEB3, (b) BEB4, (c) BEB5 and (d) BEB6. BNR domains are denoted by green bars and the regions that belong to EGF domains are in pink. Yellow bars highlight loops that are protected by an SS3 type disulfide bond, which is common for all BEBs in RELN. Red and orange sites are residues within 3.5 Å from the Zn2+ and Ca2+ ions, respectively (a list is in Table S1). Red dotted lines along the abscissa highlight regions that are connected by disulfide bonds and blue arrows point to sites containing a cysteine which does not create any disulfide bond. Residues that interact with ApoER2 receptors within 4 Å radius are indicated by cyan stars (based on PDB ID 5b4x, data in Table S1).

Fig 3: Cross-correlation of BEBs structures in MD simulations. Cross-correlation heatmaps for individual BEB structures: (a) BEB3, (b) BEB4, (c) BEB5 and (d) BEB6 show which regions tend to move in the same (correlated, colored in red) or in the opposite (anti-correlated, colored in blue) directions with respect to each other in the global modes. (e) The crystal structure of BEB5 fragment colored by values obtained from cross-correlation analysis for the residue K2194 which is located in the black box region (a black arrow denotes that residue). Ca2+ interface residues are displayed as orange sticks, those for Zn2+ in red sticks, and ApoER2 binding site is colored in cyan. A loop protected by SS3 (displayed in Fig. 1c) is denoted by a yellow shadow. Disulfide bonds are shown as thin red sticks and C2101, which serves for RELN dimerization, is denoted by blue spheres.

Fig 4: Multidomain structure of RELN. (a) Full structure of RELN in a schematic, multidomain representation and its proteolytic fragments after N- and C-terminal processing by ADAMTS-4 (P1244-A2688) are displayed. Green rectangles represent BNR domains, and EGF domains are colored in dark pink. RELN contains eight subunits, which consist of BNR-EGF-BNR domains (BEB). The blue box denotes the central part of RELN that was studied in the paper. Red dots show Zn2+ binding sites and yellow crosses represent cysteines that do not form a disulfide bond. C2101 located in BEB5 is responsible for RELN homodimerization. The enzymatically decisive serine residue in the GKS1283D sequence is indicated by a blue rectangle. The crystal structure in the inset shows the 3-D structure shared by BEB modules with all possible disulfide bonds displayed: (i) disulfide bonds within an EGF domain (named SS), (ii) disulfide bonds that zip BNR domains (SS1 in subunit BNR-A and SS2 in subunit BNR-B), (iii) a disulfide bond that keeps together a loop comprised of residues 8–12 (SS3), and (iv) a disulfide bond that links BNR11 with BNR12 (SS0) which is characteristic to BEB6 only. Close views of Zn2+ binding sites in (b) BEB6 and (c) BEB5 are displayed in the insets. Residues which coordinate Zn2+ are shown: H2061, H2074, E2264 (in BEB5) and E2397, E2399, H2460 (in BEB6). The yellow fragment of the protein (whose sequence is displayed) corresponds to the loop located in vicinity of the Zn2+ binding site and protected by SS3 in both BEB5 and BEB6 modules. The SS0 bond (orange) is also displayed in BEB6.