Structural comparison of the VWF C4 domain with other known VWC structures. (A) Sequence alignment of VWF C4, Col2a (PDB entry 5NIR), CCN3 (PDB 5NB8), and CV-2 (PDB 3NK3) with 3 stretches of structurally aligned residues using PDBeFold³⁶  marked in boxes. The remaining residue stretches were manually aligned, using the conserved cysteine pattern as a guideline. The VWF C4 domain residue numbers and secondary structural elements are shown on top of the alignment. Disulfide bridges are highlighted in different colors, demonstrating the different levels of positional conservation among the different VWC sequences numbered as in Figure 1C and Table 2. VWF residues that are shown in the structure presentation of the C4 domain (Figure 1C) are highlighted in corresponding colors. (B) Schematic representations of distinct disulfide patterns in VWF C4 (left) and Col2a/CCN3/CV-2 (right), using the same colors as in panel A. (C) Superimposition of all 4 VWC domain structures aligned to SD1, showing a considerably more kinked SD1/SD2 arrangement in the lowest energy VWF C4 conformer (compare Figure 1C) than those observed for the other 3 VWF domain structures (left). Inset zooming into the SD1/SD2 interface, demonstrating structural diversity of the interdomain disulfide bridges 2 and 4. For visual clarity, the zoomed-in disulfide pattern of Col2a, which is closely related to that of CCN3, is not shown. When viewed along SD1, the twist angle of CCN3 is the most distinct from those observed for VWF C4, Col2a, and CV-2 (right). (D) Schematic representation of twist angles calculated for representative VWC domain structures (panel C, right). (E) Twist/tilt angle analysis of VWC structures. Angle clusters of crystal structures with multiple copies are highlighted with circles in corresponding colors. The lowest energy conformation of the VWF C4 domain used in other figures is highlighted in yellow.