Figure 1: Overall structure of Net4.

(a) Ribbon model of Net4-ΔC viewed in the ‘open face’ orientation. The domains LN (teal), LE1 (orange) LE2 (magenta) and LE3-3¹/₂ (green) are coloured accordingly. N-linked glycans are drawn as red sticks, disulfide bridges are drawn as yellow sticks, and the calcium ion as a red sphere. (b) Secondary structure diagrams. Disulfide connectivity in domains LN and LE are indicated by yellow dotted lines, visible N-linked glycans by green stars with the Asn residues N⁵⁶, N¹⁶³ and N³⁵³ highlighted. β-strands are depicted as arrows and the α-helices as cylinders, each labelled with the first and last residue. The individual loop segments are shown in different colour codes. (c) Interface between domains LE1 and LN. The KAPG motif is free in solution and not visible in the structure. (d) Secondary interface between domains LE1 and LN. The loop d segment between Cys307 and Cys329 of subdomain LE1 is in contact with the N-terminus and the loop S5-H3 of domain LN. (e) Complex of Net1 (marine; PDB 4OVE) and DCC (orange; PDB 4URT) at site 1 with interacting residues shown at full opacity and the same complex with Net4 (green) replacing Net1. (f) Complex of Net1 (marine) and DCC (orange; PDB 4URT) at site 2 with interacting residues shown at full opacity and the same complex with Net4 (green) replacing Net. (g) Complex of Net1 (marine) and neogenin (pink; PDB 4PLN) at site 1 with interacting residues shown at full opacity and the same complex with Net4 (green) replacing Net1. Similar to site 1 of the DCC-Net1 complex, F441 (red) of Net4 occludes Met959 of neogenin from the hydrophobic pocket. L456 (red) of Net4 again clashes with the binding partner. (h) Net1 (marine) in complex with neogenin (pink) at site 2. Note the difference in the surface charge at the neogenin binding site (indicated by a box) of Net1 (left) and the corresponding site on Net4 (right).