Structural basis of von Willebrand factor multimerization and tubular storage

Abstract

The von Willebrand factor (VWF) propeptide (domains D1D2), is essential for the assembly of VWF multimers and its tubular storage in Weibel-Palade bodies. However, detailed molecular mechanism underlying this propeptide dependence is unclear. Here we prepared Weibel-Palade body-like tubules using the N-terminal fragment of VWF and solved the cryo-EM structures of the tubule at atomic resolution. Detailed structural and biochemical analysis indicate that the propeptide forms a homodimer at acidic pH through the D2:D2 binding interface and then recruits two D'D3 domains forming an intertwined D1D2D'D3 homodimer in essence. Stacking of these homodimers by the intermolecular D1:D2 interfaces brings two D3 domains face-to-face and facilitates their disulfide linkages and multimerization of VWF. Sequential stacking of these homodimers leads to a right-hand-helical tubule for VWF storage. The clinically identified VWF mutations in the propeptide disrupted different steps of the assembling process leading to diminished VWF multimers in von Willebrand diseases (VWD). Overall, these results indicate that the propeptide serves as a pH sensing template for VWF multimerization and tubular storage. This sheds light on delivering normal propeptide as template to rectify the defects in multimerization of VWD mutants.

Assembly of von Willebrand Factor Tubules within VivoHelical Parameters Requires A1 Domain Insertion

“…One possible explanation would be that A1 packs against the outside of the D-assembly tubule, stabilizing its structure. We found, however, that A1 directly inserts into the tubule, explaining the discrepancy between the geometries of in vitro [12][13][14] vs. in vivo 15 VWF tubules.…”

“…Notably, with the A1 domain present, it was possible to obtain a map at a nominal resolution of 3.2 Å (supplemental Figure 1) for a pair of beads interacting between helical turns, whereas previous local refinements of such bead pairs yielded maps to only about 4.5 Å resolution. 13,14 The presence of the A1 domain within the tubule explains the difference in helical parameters between the in vitro tubules generated from D assemblies and the full-length tubules studied in cells (Table 1). Fourier analysis of the tubules in WPBs showed an axial rise per subunit of 27.7 Å.…”

“…However, the A1 domain did not merely dock onto the D-assembly tubule but rather replaced the interhelical contacts observed previously. 13,14 In the structure of the D-assembly tubule, these contacts occurred by reciprocal interaction of the C8-1/TIL1 regions. 13 In the tubules containing A1, the A1 domain was found wedged between the E1 module in one bead and the C8-1 region of another bead (Figure 3B).…”

“…12 The structure of these tubules has been determined, first at low resolution 12 and then at higher resolution using cryo-electron microscopy (cryo-EM). 13,14 In addition, the organization of VWF in intracellular WPBs was studied by cryo-EM tomography and Fourier analysis. 15 In all these cases, and in structural studies of filaments formed by the amino-terminal region of the intestinal mucin, MUC2, 16 which is homologous to VWF, the fundamental unit of the supramolecular assembly was a globular “bead.” MUC2 filaments are made of linear strings of these beads, 16 whereas the VWF tubule is made of successive beads spiraling around a central axis.…”

Protein Markers Related to Non-halal Slaughtering Process of Rat as Mammal Animal’s Model Detected Using Mass Spectrometry Proteome Analysis

“…Meanwhile, the Von Willebrand factor was a protein involved in the response to stimulus. This protein worked in the blood coagulation process caused by wounds or injuries on the blood vessels [33]. The process of cervical dislocation could be thought to stimulate the excretion of the Von Willebrand factor due to the occurrence of injuries in an animal body.…”