β-Strands are numbered according to the initial bovine aSFP CUB domain structure  (light blue). The front β-sheet comprises strands 1, 3, 10, 5 and 8. The cbCUB1 of human C1s  (red) lacks the first two β-strands and harbours a Ca2+ ion (orange sphere) bound at the interface between loops L5 and L9, on the side opposite to the C-terminus (Ct). Nt, N-terminus. An interactive three-dimensional structure of Figure 1 is available at http://www.BiochemJ.org/bj/439/0185/bj4390185add.htm.
The set of sequences shown includes both experimentally determined (see Table 1) and predicted cbCUBs. The average boundaries of β-sheets and loops are shown at the top. Conserved residues defining the generic CUB domain signature and cysteines are coloured light blue. The tyrosine residue conserved in the cbCUB subset is coloured purple, and the acidic residues involved in Ca2+ coordination are coloured orange (monodentate ligands) and red (bidentate ligand). Residues contributing additional Ca2+ ligands, mostly through their carbonyl group, are shown in yellow. cbCUB domains identified in Branchiostoma belcheri (BRMASP_1 and BRMASP_2), Nematostella vectensis (NVMASP_1, NVMASP_2, NVTLL_1 and NVTLL_2) and Aciduliprofundum boonei (B5IAQ4) are highlighted in grey. All other sequences are of human origin. ATRN, attractin; GP126, G-protein-coupled receptor 126; L, loop. The level of residue conservation is indicated at the bottom of the sequence alignment as follows: strictly identical (*), strongly similar (:) and similar (.).
Figure 3Ca2+ coordination in cbCUB domains and other Ca2+-binding sites
Two representative examples of the Ca2+ coordination observed in the cbCUB family are shown: MASP1 CUB2 (A) and human NRP2 (B). For comparison purposes, a classical EF hand, an LA module and a cbEGF are displayed in (C–E) respectively. Binding of an Mg2+ ion to C1s cbCUB1 is shown in (F). The Ca2+ ion is shown in green and Mg2+ is in magenta. The water-coordinating ligands are coloured cyan. The Ca2+ ligands lying approximately in the same plane are indicated by orange broken lines, whereas the two ligands occupying the summits of the pyramid on an axis perpendicular to this plane are shown by grey broken lines. As in Figure 2, bidentate ligands are shown in red, monodentate side-chain ligands in orange and residues providing main-chain carbonyl ligands in yellow.
Figure 4Ca2+-induced conformational changes at the tip of cbCUBs
(A) Stable compact conformation of the tip of human MASP2 CUB1 (Ca2+-bound). (B) Partly disordered conformation of the corresponding part of rat MASP2 CUB1 (Ca2+-free). The tip of loop L9 (residues 103–106) is totally disordered. A different network of stabilizing interactions is found in the two cases. The side chains of residues at the top of loops L5 and L9 [Tyr59(Tyr55), Asp105(Asp101) and Tyr106(Tyr102)] have different orientations and their functional implications are described in Figure 5. The conserved Tyr24(Tyr20) residue (magenta) stabilizes the position of loop L3 by interaction with the acidic side chain of Asp60(Asp56) and the main chain of Glu52(Glu48) in both cases.
(A and B) Multi-point cbCUB-mediated interactions are highlighted in MASP1  and CUBN . (C–F) Details of these interactions for MASP1 CUB1 (C) and CUB2 (E), cubilin CUB6 (D) and CUB8 (F). (G) Residues contributed by loops L5 and L9 involved in protein–protein interactions. In (D), (F) and (G), CUBN residues shown to interact with the gastric intrinsic factor  are highlighted in blue. The central electrostatic interactions are shown by broken lines in (D) and (F). In (C), (E) and (G), residues whose mutation abolishes or weakens the interaction of MASP1 with its cognate recognition proteins MBL and the ficolins are shown in red and orange respectively. The same colour code is used for mutations in C1r, C1s and MASP2 [6,11] in (G). Mutations at Glu52 and Ser107 in human MASP2 correspond to unpublished data obtained by N. Thielens. In (G), the conserved residues corresponding to the CUB domain signature are shown in light blue and the three acidic Ca2+ ligands are denoted by red asterisks. Ca2+ ions and water molecules are represented as yellow and blue spheres respectively. Ionic bonds involved in Ca2+ coordination are not depicted. The level of residue conservation is indicated at the bottom of the sequence alignment as follows: strictly identical (*), strongly similar (:) and similar (.). Water was not observed in (D) and (F) due to the low resolution .
Figure 6Similar electrostatic interactions mediated by acidic Ca2+ ligands of LA and cbEGF domains
(A and B) Overall and close-up views of the interaction between LA modules 3 and 4 of LDLR and RAP . (C and D) Overall and closer views of the interaction between LA module 1 of ApoER2 and reelin . (E and F) Overall and close-up views of the interaction between module EGF1 of LDLR and PCSK9 . Ca2+-coordinating residues are shown in orange, and the basic lysine or arginine residue involved in the ionic interaction is shown in pink.
Figure 7Some of the diverse modular proteins harbouring cbCUBs
N-terminal ends are on the left-hand side in all cases. Transmembrane domains are represented as black boxes. Signal sequences and cytoplasmic domains are not depicted. The domain nomenclature and symbols used are those defined in the SMART database .