Structures of VWF tubules before and after concatemerization reveal a mechanism of disulfide bond exchange

Abstract

von Willebrand factor (VWF) is an adhesive glycoprotein that circulates in the blood as disulfide-linked concatemers and functions in primary hemostasis. The loss of long VWF concatemers is associated with the excessive bleeding of type 2A von Willebrand disease (VWD). Formation of the disulfide bonds that concatemerize VWF requires VWF to self-associate into helical tubules, yet how the helical tubules template intermolecular disulfide bonds is not known. Here, we report electron cryomicroscopy (cryo-EM) structures of VWF tubules before and after intermolecular disulfide bond formation. The structures provide evidence that VWF tubulates through a charge-neutralization mechanism and that the A1 domain enhances tubule length by crosslinking successive helical turns. In addition, the structures reveal disulfide states before and after disulfide bond-mediated concatemerization. The structures and proposed assembly mechanism provide a foundation to rationalize VWD-causing mutations.

Graphical abstract

Figure 1.

Structure of a VWF tubule. (A) Schematic of the domain organization of the VWF proprotein. Each D assembly except D′ is made of domains VWF type D domain, C8, TIL (trypsin inhibitor-like cysteine-rich domain), and E. D1 and D2 form a prodomain that is cleaved by furin protease before secretion. (B) Strategy to obtain VWF monomers and disulfide-linked dimers. VWF D1-A1 was expressed in Expi293 cells and purified from the media as monomers and disulfide-linked dimers. The furin cleavage site was mutated to ASAS to prevent prodomain dissociation. (C) DLS experiments of purified D1-A1 incubated at indicated pH showing an increase in average hydrodynamic radius with a decrease in pH. Error bars represent the difference between the means from 2 replicates. (D) Representative micrographs from negative-stain electron microscopy showing VWF dimers incubated at pH 7.4 and 5.2 (scale bars, 50 nm). (E) Quantification of (D) showing particle lengths measured from 3 micrographs from samples incubated together overnight. The bottom, middle, and top lines of the shaded boxes represent the first quartile, the median, and the third quartile of measured particle length, respectively. The length of the whiskers below and above the box plot represent the lesser of the range of data or 1.5 multiplied by the interquartile range. (F) Schematic and cryo-EM structure of the dimer-derived tubule showing the bead-like arrangement. The structure forms a right-handed helix with a helical rise of 26.8 Å and a helical twist of 83.3°. The cryo-EM map has been Gaussian filtered for visualization.

Figure 2.

Organization of VWF within a tubule. (A) Cryo-EM map of the dimer-derived VWF tubule in gray with a single bead colored by each of the 4 monomeric VWF subunits from which it is formed. Beads have twofold symmetry. (B) A single bead. The left view has the same orientation as in (A) and has a red dashed line separating symmetrical halves. The right view shows the luminal facing portion of the bead, which is formed by 2 antiparallel D1D2 prodomains that form a cradle for the binding of 2 juxtaposed D′D3 domains. (C) A VWF monomer, colored by domain, adopts an extended conformation that spans 2 beads. The D1D2 prodomain is in 1 bead, and the D′D3 and A1 domains are in a neighboring bead. Domains of neighboring VWF molecules are colored gray and are shown with thinner loops, helices, and strands.

Figure 3.

The A1 domain is a component of the tubule and links helical repeats. (A) Cryo-EM map of the dimer-derived VWF tubule in gray with A1 domains in pink. A single A1 domain is boxed together with the domains of the neighboring molecules with which it interacts. (B) Details of the trans interactions an A1 domain makes with 2 neighboring VWF molecules. Distinct molecules are denoted by no apostrophe, 1 apostrophe (’), or 2 apostrophes (’’). (C) Quantification of tubule length observed by negative-stain electron microscopy with and without the A1 domain. Particle lengths were measured from 3 micrographs from samples incubated together overnight. Whisker plots are as described in the Figure 1E legend.

Figure 4.

Structural basis for pH-dependent VWF tubule formation. (A) The Cα positions of the 33 histidine residues resolved in the structure are depicted as spheres color-coded based on sequence conservation obtained from 140 homologs using the ConSurf server. (B) Details of the intramonomer salt bridge made by H395. (C) Details of the intramonomer salt bridge made by H817. (D) Interface between D2-D3 of one molecule (denoted D2-1 and D3-1; gray) and the D2 domain of another molecule (D2-2; teal) (top). The interfaces are separated and colored by electrostatic potential calculated at pH 7.4 (middle) and 5.2 (bottom). Electrostatic potentials were calculated using default coulombic parameters in ChimeraX 1.3. The positions of surface-exposed histidine residues are marked with a black circle or a yellow star if highlighted in panels (E-G). (E-G) Examples of histidine residues—(E) H421, (F) H556 and H817, and (G) H460—that change protonation state upon pH change from 7.4 to 5.2 in electronegative local environments.

Figure 5.

VWF concatemerization proceeds through a disulfide exchange mechanism. (A) Overview showing the juxtaposition of domains D′-A1 from separate molecules in the center of a single bead. (B-C) Arrangement of intra- and intermolecular disulfide bonds in VWF tubules generated with dimeric D1-A1. (D-E) Arrangement of interfacial cysteines and intramolecular disulfide in VWF tubules generated with monomeric D1-A1. (F) Superposition of D3 domains showing the rearrangement of the 1091-1097 loop between monomer and dimer states. Model superposition was performed using the matchmake function of ChimeraX.

Figure 6.

Type 2A mutations. (A) Positions of VWD type 2A missense mutations mapped onto the structure of VWF D1-A1 as it occurs in a tubule. (B) Y87 lines a crevice in the VWD1 domain. The type 2A VWD mutation Y87S would allow solvent into this crevice, potentially disrupting tubule formation. (C) R202 forms intra- and intermolecular interactions with 2 additional VWF molecules denoted in purple and dark teal. R202W, a mutation causing type 2A VWD, would disrupt these interactions. A dashed green line shows a π–cation bond. (D) A1 domain in light pink with residues altered in type 2A VWD colored in dark pink. Leucine residues described in the text are labeled.

Figure 7.

Model for VWF tubule assembly. (A) Options I-IV show the possible molecules that could be disulfide bonded at their C-termini in native tubules generated with full-length VWF. Distances between A1 domains (calculated between A1464 Cα positions) are shown below, together with surface representations of potential C-terminally–linked dimers and their quantified interfacial surface area. An overview is provided with a single bead denoted with a dashed white outline. Interfacial surface areas were calculated using PDBePISA. (B) Schematic showing a hypothetical maturation pathway for full-length VWF based on our cryo-EM structures and prior work. In this model, the acidic environment of the Golgi induces 2 changes in the VWF dimer: the A2-CK domains zipper together, and the N-terminal heads form a “dimer swapped” conformation with D3 of one monomer nestling in a D1-D2 cradle of the other monomer. N-terminal dimer association leads to tubule assembly. Within the tubule, D3 domains from different dimers are juxtaposed. Once positioned, a disulfide exchange occurs where C1097 is liberated from an intramolecular disulfide bond to form 1 of 2 disulfides that concatemerize VWF dimers at their D3 domain. The second disulfide, between adjacent C1142 residues, occurs without disulfide exchange. With dimers linked at their D3 domains, the VWF tubule can unfurl into a high molecular weight VWF concatemer upon exocytosis into the blood plasma.