Modeling and docking
The N-terminal region of VWF was structurally modeled to investigate the structural-functional impact of our mutations. The D1 and D2 domains were modeled independently on the ITASSER threading server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) while assigning disulfide-bonded cysteine “У” sulfur atom restraints, based on previously suggested disulfide combinations in these domains . The model with the best C-score from each domain was chosen as its final structure. The crystal structure of the D´-D3 assembly was downloaded from the structure database (PDB ID: 6N29; Resolution: 2.5 Å) .
The joined model of the VWFpp (D1 and D2 domains) and the D´-D3 assembly was generated using overlapping inter-domain linker regions. The linked model was subjected to a brief structure refinement simulation based on previously reported methodology . The lowest energy structure in the refinement simulation trajectory was chosen for further applications. The model´s dimensions and orientation were investigated in the context of its ability to form symmetric multimers. All visualization, image rendering, and structural analysis were performed on the YASARA and Chimera platforms .
Consequently, the generated model of D1D2D´D3 was used to dock two proteins Sec24 and ARF1, for whom limited evidence exists for their role in anterograde transport of VWF. The atomic structure of ARF1 and Sec24 was extracted from the PDB ID: 2J59 with resolution: 2.1 Å and PDB ID: 3EFO with resolution: 2.7 Å, respectively . All water molecules and heteroatoms including GTP, as well other molecules like peptides were removed from both structures. Both structures were refined using a previously tested simulation protocol before implementing docking onto the VWFpp model. In the case of Sec24, since the original structure is a complex of Sec23/Sec24, only the PDB coordinates of the recruitment protein i.e. Sec24 from the refinement simulation were used for docking. Similarly for PDB ID: 2J59, which is a complex of ARF1/ARHGAP21, only the co-ordinates of AERF1 were used for docking. The docking simulation was performedon the SwarmDock server (https://bmm.crick.ac.uk/~svc-bmm-swarmdock/submit.cgi) using the default conditions of the server . The highest scoring docking poses (n=10) from the first ten clusters based on hierarchical clustering with the democratic scoring scheme were diligently investigated especially with respect to the reported mutated residues.