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.