An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution

J Mol Biol. 2021 Jun 25;433(13):166954. doi: 10.1016/j.jmb.2021.166954. Epub 2021 Mar 24.


Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.

Keywords: biophysical techniques; blood coagulation; metallopeptidase; platelet aggregation; protein–protein interactions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADAMTS13 Protein / metabolism*
  • Cross-Linking Reagents / chemistry
  • Humans
  • Kinetics
  • Models, Molecular
  • Peptides / chemistry
  • Protein Binding
  • Protein Processing, Post-Translational*
  • Solutions
  • von Willebrand Factor / chemistry*
  • von Willebrand Factor / isolation & purification
  • von Willebrand Factor / metabolism*


  • Cross-Linking Reagents
  • Peptides
  • Solutions
  • von Willebrand Factor
  • ADAMTS13 Protein