Key residues regulating von Willebrand factor A1/A2 interactions: insights from molecular dynamics simulations and...

Von Willebrand factor (VWF), secreted by endothelial cells and megakaryocytes, plays a critical role in hemostasis. Under normal physiological conditions, VWF adopts a globular conformation in which the glycoprotein Ibα-binding site on A1 domain is occluded by the adjacent A2 domain. Studies indicate that exogenous free A2 fragments can improve coagulation function following traumatic brain injury by competitively binding to endogenous VWF-A1, highlighting the therapeutic potential of targeting A1/A2 interaction.

This study aimed to identify key residues regulating the A1/A2 interaction at the interface.

In this study, we used the HADDOCK platform to obtain the optimal conformation of A1/A2 complex and used molecular dynamics simulations to map key binding sites. We conducted multiscale validation using steered molecular dynamics, biolayer interferometry, and atomic force microscopy.

Our results identified 3 pairs of strong interdomain hydrogen bonds at the A1/A2 interface: R1334-E1598, Q1367-V1546, and D1323-R1575. Computational analysis indicated that mutations in both A1_mut (D1323A/R1334A/Q1367A) and A2_mut (V1546A/R1575A/E1598A) reduced the A1/A2-binding affinity and conformational stability. Biolayer interferometry assays confirmed a marked reduction in affinity for A1_mut, while the atomic force microscopy experiment showed only a nonsignificant reduction trend. In contrast, A2_mut significantly disrupted A1/A2 binding in both assays.

By integrating computational prediction with experimental validation, we identified residues D1323, R1334, and Q1367 on A1, along with V1546, R1575, and E1598 on A2, as key residues that maintain the A1/A2 interaction. These findings enhance our understanding of the VWF-A1/A2 interaction mechanism and provide novel molecular targets and a theoretical basis for developing targeted therapies for associated disorders.