Protein superglue that holds VWF together

In this issue of Blood, Zhou and Springer report on the structure determination of the von Willebrand factor (VWF) C-terminal cysteine knot domain (CTCK), finally uncovering the structural basis for the super-stable CTCK dimer that underpins platelet adhesion.¹ 

VWF, a primary blood clotting protein, forms ultralarge multimers with molecular weights exceeding 2000 kDa. This factor serves as a docking station or molecular bus²  to which numerous proteins bind during the formation of blood clots. How such enormous structures are able to withstand the high pressure and velocity environment of the circulatory system has long been a mystery.

Acting like the molecular equivalent of superglue, CTCK mediates the formation of the VWF dimer, the fundamental building block of the functional, ultralarge VWF multimers (see figure). VWF deficiency and/or dysfunction (von Willebrand disease) is associated with various blood-related diseases and physiological conditions,³  particularly bleeding which is most apparent in tissues having high blood flow shear in narrow vessels. Previously, electron microscopy was used to establish an intertwined bouquet structure of VWF which resembles the elongated stem of a flower.^4,5  But this picture raised a fundamental question: how could such an elongated structure possibly survive the intense shearing mechanical forces, as well as the biochemical “forces” of disulfide reduction and proteolysis, present in the circulatory system?

The CTCK structure by Zhou and Springer¹  answers this question in spades. Despite its diminutive size, CTCK possesses the ability to self-dimerize with a strength that surely has few peers in the protein universe. The “triple reinforcement” (cysteine knot in the monomer, intermolecular β-sheet formation, and intermolecular disulfide bridges in the dimer) is at the heart of this ultrastability. In a very small local area, multiple intra- and intermolecular disulfides are clustered together to boost the strength of both monomer and dimer structures: the intramolecular ones help to ensure that each monomer remains stable and resistant to protein unfolding, while the buried nature of the intermolecular disulfides is naturally resistant to disulfide reduction. Moreover, super β-sheet (ie, intermolecular β-sheet) formation is widely recognized as one of the “strongest” linking mechanisms between monomers. Additionally, numerous favorable side-chain interactions and the burial of proportionately large surface area (close to one-third of the entire monomer surface) all contribute to the ultrastability of the CTCK dimer (see figure). This dimer structure is truly a “beauty.”

From a technical perspective, the structure determination of CTCK was no small feat because of complications such as glycosylation and proper disulfide formation. Successful protein expression was achieved using mammalian cells, a complex task in itself. Although protein production in HEK293 cells is not amendable to routine selenomethionine labeling for standard crystallographic phasing, fortunately, the successful crystallization condition contained zinc metal which served as a heavy atom for solving the phase problem. Furthermore, the unusually high solvent content (88%) greatly helped improve the map quality which would otherwise have been challenging for such low-resolution data.

As with most determined structures, the structure determined by Zhou and Springer¹  provides immediate insight into the structural mechanisms involved with VWF. Not only does it provide direct knowledge of the VWF dimer binding mechanisms, but it also offers clear structural rationale for CTCK point mutations implicated in von Willebrand disease: they all weaken this dimer interaction. Moreover, it is also anticipated that this structure will help facilitate the characterization of related homologs involved in numerous cellular processes. However, like many other structures, the CTCK structure also raises some interesting questions. For example, the CTCK dimer structure seems incompatible with the fact that shear stress can provoke a rearrangement of disulfide bridges in the C-terminal part of the protein,^6,7  a process that contributes to the incorporation of new VWF molecules into the assembled VWF multimers at the vascular surface. Based on the crystal structure, it is difficult to comprehend how some sort of disulfide bridge swapping could be accomplished without greatly compromising the triple reinforcement. Clearly, more studies are needed to gain a better understanding of the complex and multifaceted VWF molecule. From a clinical perspective, although the CTCK structure is a big step forward in our basic understanding of the dimer formation, developing any potential therapeutic benefits from this structure is still likely a fair ways off. The challenge here will be to “outsmart” Mother Nature and design either (1) an even stronger dimer that is less compromised by disease-causing mutations (though the challenges of delivering a genetic fix are still formidable) or (2) finding novel compounds that can enhance CTCK dimer formation for those with deleterious point mutations.

This structure serves as a poignant example of the capacity for evolution to engineer exquisite solutions to highly specific problems, in this case marshalling an array of structural components that make the CTCK assembly as stable/destruction resistant as possible. In essence, CTCK acts like protein superglue that holds VWF together. Indeed, this structure is akin to highly-reinforced concrete blocks strengthened not only by standard steel bars but also by polymers and/or alternate composite material.