# In-Silico Characterization of von Willebrand Factor Bound to FVIII
## 3 Results
### Interface Analysis  
Since binding free energy prediction with PyDock and PRODIGY, generally, provides an indication of binding driving forces, we further focused our calculations on specific protein-protein interface descriptors. We analyzed the protein-protein interfaces of VWF/FVIII complexes: B domain-deleted FVIII (Table 3), secreted full-length FVIII (Table 4), and etanesococog alfa (Table 5). Two interfaces were analyzed: (i) one that involves the heavy and the light chains interactions; (ii) and the VWF/FVIII interface. BDD-FVIII and the FL-FVIII showed similar heavy/light chain interfaces (Tables 3 and 4) in terms of \(\Delta\Delta\)Gsolv, which was slightly lower in the VWF/FL-FVIII (more favorable) compared to BDD-FVIII complex. These results are coherent with PyDock and Prodigy predictions. Furthermore, according to prediction of binding free energy (PRODIGY calculations) the interactions in the VWF/FVIII interface of the etanesococog alfa were characterized by the most favorable (lowest) predicted free energy \(\Delta\)AGsolv, kcal/mol (Table 5), compared to the other analyzed complexes. This lower \(\Delta\Delta\)Gsolv, kcal/mol (more favorable) corresponded to higher \(\Delta\Delta\)Gsolv, kcal/mol at interface between heavy and light chains in etanesococog alfa, compared to other analyzed complexes. Therefore, it is likely that more stable interactions between VWF/FVIII correspond to a destabilization of FVIII heavy/light chain interface. The results reported in Tables 3-5 indicated that the etanesococog alfa is the most stable VWF/FVIII complex, in terms of specific stability of monomers and interface interaction, compared to the VWF/BDD-FVIII and VWF/FL-FVIII complexes, according to findings of [PERSON] et al. [13]. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Complex** & **FVIII-Interacting Domains** & **AGbinding (kcal/mol)** & **pKd** \\ \hline BDD-FVIII & A3-C1 light chain & \(-15.3\) & \(11.3\) \\ \hline Efanesococog alfa (BIVV001)–atomic model (PDB: 7 KWO.) & A3-C1 light chain n & \(-19.5\) & \(14.3\) \\ \hline Secreted full-length FVIII & B domain–heavy chain & \(-14.1\) & \(10.4\) \\ \hline \hline \end{tabular}
\end{table} 
Table 2: PRODIGY rescoring of PYDOCK-predicted complexes. \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Protein Residues** & **Interface Residues** & **ASA**, \(\hat{\mathbf{A}}^{2}\) & \(\Delta\)**Gsolv, kcal/mol (Per Residue)** \\ \hline
**MON H (heavy chain)** & 693 & 681 & 38,466.4 & \(-608.4\) (\(-0.88\)) \\
**MON L (light chain)** & 644 & 624 & 34,017.4 & \(-570.1\) (\(-0.89\)) \\
**MON V (ligand VWF)** & 428 & 406 & 23,642.1 & \(-370.7\) (\(-0.87\)) \\ \hline \hline \multicolumn{4}{c}{**INTERFACE H-L**} \\ \hline
**INTERACTING RESIDUES, MON H** & & 90 & \\
**INTERACTING RESIDUES, MON L** & & 100 & \\
**INTERFACE AREA, \(\hat{\mathbf{A}}^{2}\) (per interacting residue)** & & 3322.2 (17.50) & \\ \(\Delta\)**AGsolv, kcal/mol (per interacting residue)** & & \(-42.7\) (\(-0.22\)) & \\ \hline \hline \multicolumn{4}{c}{**INTERFACE L-V**} \\ \hline
**INTERACTING RESIDUES, MON L** & & 42 & \\
**INTERACTING RESIDUES, MON V** & & 57 & \\
**INTERFACE AREA, \(\hat{\mathbf{A}}^{2}\) (per interacting residue)** & & 1742.1 (17.60) & \\ \(\Delta\)**AGsolv, kcal/mol (per interacting residue)** & & \(-13.3\) (\(-0.13\)) & \\ \hline \hline \end{tabular}
\end{table} 
Table 3: PISA results for the VWF/BDD-FVIII complex. Values within brackets refer to the value specific per residue. Accessible surface area (ASA) of interface (\(\hat{\mathbf{A}}^{2}\)). To obtain further insight in the protein-protein interactions, we carried out PCN analysis of the three complexes: VWF/BDD-FVIII, \(\mathrm{\acute{e}anesoctocog\ alfa}\), and \(\mathrm{VWF/FL-FVIII}\). Results are shown in Table 6, according to the description provided in Materials and Methods. PCN analysis (Table 6) has shown that the interface roughness, (Q/R) was very similar in analyzed FVIII structure models.