Structural specializations of A2, a force-sensing domain in the ultralarge vascular protein von Willebrand factor

Abstract

The lengths of von Willebrand factor (VWF) concatamers correlate with hemostatic potency. After secretion in plasma, length is regulated by hydrodynamic shear force-dependent unfolding of the A2 domain, which is then cleaved by a specific protease. The 1.9-A crystal structure of the A2 domain demonstrates evolutionary adaptations to this shear sensor function. Unique among VWF A (VWA) domains, A2 contains a loop in place of the alpha4 helix, and a cis-proline. The central beta4-strand is poorly packed, with multiple side-chain rotamers. The Tyr-Met cleavage site is buried in the beta4-strand in the central hydrophobic core, and the Tyr structurally links to the C-terminal alpha6-helix. The alpha6-helix ends in 2 Cys residues that are linked by an unusual vicinal disulfide bond that is buried in a hydrophobic pocket. These features may narrow the force range over which unfolding occurs and may also slow refolding. Von Willebrand disease mutations, which presumably lower the force at which A2 unfolds, are illuminated by the structure.

Fig. 1.

The VWF A2 domain. (A) Overall structure of mature VWF. Cysteines are shown as vertical lines and are connected for chemically defined disulfides (10). N- and O- linked glycans are closed and open lollipops, respectively. (B and C) Ribbon diagram of VWF A2 domain. N-linked glycosylation sites are shown in stick. The C-terminal disulfide bond and ADAMTS13 cleavage site residues (Y, Y1605; M, M1606) are shown as sticks. In C, the α4 helices in VWF A1 and A3 are shown after superposition of the domains. (D) Cα B-factors. Higher B-factors are represented by a thicker chain-trace and a spectral shift from blue to red. (E) Structure-based sequence alignment. β-Strands and α-helices are colored. Dots show decadal residues. The ADAMTS13 cleavage site is indicated by an arrow. Glycosylation sites are indicated by asterisks. Residues involved in VWD mutations are highlighted in yellow. Unaligned residues are lower case and residues absent in the A2 structure are italicized.

Fig. 2.

The α4-less loop environment. (A) The α3 and α5-helix dipole moments are symbolized. (B) The Asp-1614 N-cap. (C) The Arg¹⁶¹⁸ C-cap and H-bond to the cis-peptide. Key side chains and helix main chain (in B and C) are shown in stick, and H-bonds are dashed.

Fig. 3.

Structural specializations of A2. (A and B) The distinct rotamers of Leu¹⁶⁰³ (A) and Met¹⁶⁰⁶ (B). (C) The 8-membered ring formed by the vicinal disulfide. (D) The vicinal disulfide and its hydrophobic pocket. Key side chains are shown in stick and water molecules as spheres. (E) A water in a largely hydrophobic environment, with both Ser¹⁵¹⁷ rotamers shown. 2Fo-Fc densities around selected side chains and waters are contoured at 0.85 σ.

Fig. 4.

Type 2A VWD mutations. (A and B) The environments around Arg¹⁵⁹⁷, commonly mutated to Trp (A) and Glu¹⁶³⁸, mutated to Lys (B). Waters are spheres (only Waters 1, 2, and 3 are buried), H-bonds are dashed, and 2Fo-Fc densities are contoured at 1 σ. (C and D) VWD mutations, with each affected residue shown as a Cα sphere.