Table 1.
Results of the I-TASSER analysis concerning the 3D models of the Monomer WT (764-2191) and p.R1205X VWF variants.
Fig 1.
Best model of the 764-2191 region of WT-VWF.
The three globular regions involving a) the D’D3, b) the entangled A1-A2, and A3 domains, and c) the connecting region with the D4 domain are shown. The D’D3 domain is shown in red, the A1 in magenta, the A2 in cyan, the A3 in orange, where some regions, involving the D4 domain, whose structure was not yet solved, are shown in yellow. The side chain of R1205 is shown in palegreen. The model was obtained with the I-TASSER program, whereas the manipulation was accomplished with PyMOL.
Fig 2.
Superposition of WT-VWF (green ribbon) and p.R1205H (cyan ribbon).
The region R1392-R1399 and R1943, involved in binding to LRP1 domain IV, are shown as yellow sticks and red sticks for WT and p.R1205H VWF, respectively. The R1943 side chains of both WT (yellow sticks) and p.R1205H variant (red sticks) are also shown. Note the large topological change for the region S764-T789, likely deriving from a huge conformational flexibility (curve red arrow). The superposition and alignment of the two structures were performed using the PyMOL program.
Fig 3.
Magnification of the WT-VWF segment encompassing R1205, from the model shown in Fig 1.
Polar interactions ≤3 Å (dashed lines) between the side chain of R1205 (shown in red sticks) and the surrounding amino acids (yellow sticks) are shown. The model was obtained with I-TASSER and manipulated with PyMOL.
Fig 4.
Molecular models obtained with I-TASSER and Haddock 2.4 programs of the adduct between WT-VWF and CCR IV of LRP1.
The VWF construct is shown in cyan, whereas the CCR IV of LRP1 is shown in pink. The interacting residues of the complex, all at distances comprised between 1.57 and 3.78 Å, are listed in S2 Table.
Table 2.
Interface features of the adducts between WT and VWF variants at R1205 and LRP1 domain IV. Chain A represents VWF, whereas Chain B is LRP1 domain IV.
Table 3.
Calculated ΔG of binding of different WT monomer species and LRP1-Cluster IV with the corresponding Kd values calculated by the PRODIGY program (T = 37 °C).
Fig 5.
Molecular models obtained with I-TASSER and Haddock 2.4 programs of the adduct between p.R1205H-VWF and CCR IV of LRP1.
The VWF construct is shown in cyan, whereas the CCR IV of LRP1 is shown in pink. The interacting residues of the complex, all at distances comprised between 1.60 and 3.45 Å, are listed in S2 Table.
Fig 6.
Molecular models obtained with I-TASSER and Haddock 2.4 programs of the adduct between p.R1205C-VWF and CCR IV of LRP1.
The VWF construct is shown in cyan, whereas the CCR IV of LRP1 is shown in pink. The interacting residues of the complex, all at distances comprised between 1.58 and 3.73 Å, are listed in S2 Table.
Fig 7.
Molecular models obtained with I-TASSER and Haddock 2.4 programs of the adduct between p.R1205LVWF and CCR IV of LRP1.
The VWF construct is shown in cyan, whereas the CCR IV of LRP1 is shown in pink. The interacting residues of the complex, all at distances comprised between 1.56 and 3.87 Å, are listed in S2 Table.
Fig 8.
Molecular models obtained with I-TASSER and Haddock 2.4 programs of the adduct between p.R1205SVWF and CCR IV of LRP1.
The VWF construct is shown in cyan, whereas the CCR IV of LRP1 is shown in pink. The interacting residues of the complex, all at distances comprised between 1.59 and 2.61 Å, are listed in S2 Table.
Fig 9.
Values of ΔG of binding of the VWF forms to LRP1 as a function of VWF interface areas.
The slope of the linear regression is -0.006271 ± 0.001458 (p = 0.0231). The dashed lines represent the 95% confidence interval of the regression.
Table 4.
Calculated ΔG of binding and the corresponding Kd values for the different VWF species and the N-terminal (H1-T266) domain of platelet GpIbα. The binding parameters were calculated by the PRODIGY program at T = 37 °C.
Fig 10.
Best models of the 764-2191 region of WT-VWF and R1205H-VWF models bound to the N-terminal (1-266) domain of platelet GpIbα.
For comparison, the crystal structure of the N-terminal (1-266) region of GpIbα bound to the VWF A1 domain (pdb: 1SQ0) is also shown. The side chain of interacting residues of GpIbα is shown in warm pink, whereas those of VWF are shown as blue sticks. The models were generated by the HADDOCK program, whereas the rendering was accomplished with Pymol.
Fig 11.
The models of the complexes of the other p.R1205X variants (p.R1205C, p.R1205L, and p.R1205S) with the GpIbα domain.
In all cases, GpIbα binds to the A1-A3 region of VWF. The models of VWF(764-2191) are shown cyan, while the GpIbα molecule is shown in pink. The side chain of interacting residues of GpIbα is shown in warm pink, whereas those of VWF are shown as blue sticks. The models were generated by the HADDOCK program, whereas the rendering was accomplished with Pymol.
Fig 12.
Conformational dynamics of the VWF A1 domain α1-β2 loop upon GPIbα binding.
The region encompassing residues 1294–1336 is shown for both WT-VWF and p.R1205L variants (green in the unbound conformation and cyan in the GPIbα-bound states). In WT-VWF, binding to GPIbα induces a defined conformational rearrangement in the α1-β2 loop (residues 1309-1314), positioning key residues like K1332 and R1334 to form direct contacts with GPIbα. In contrast, the p.R1205L mutation destabilizes the α1-β2 loop, preventing it from adopting the stable, binding-competent conformation observed in the WT. This structural disruption, evident from the altered orientation of K1332 and R1334, explains the weakened affinity for the platelet receptor. Similar loop destabilization was observed for the p.R1205C variant. Images were rendered using PyMOL.
Fig 13.
Flowchart of the entire pipeline of the modeling procedure.