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.

Centre de Biochimie Structurale INSERM CNRS and Université de Montpellier 34090 Montpellier France

Department of Molecular Biology and Genetics Aarhus University Gustav Wieds Vej 10 8000 Aarhus C Denmark

Institute of Biotechnology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czechia

Institute of Microbiology of the Czech Academy of Sciences BIOCEV Prumyslova 595 252 50 Vestec Czechia

Laboratory for Viruses and Large Biological Complexes Department of Structural Biology Molecular Biology Institute of Barcelona Barcelona Science Park c Baldiri Reixac 15 21 08028 Barcelona Catalonia Spain

Proteolysis Laboratory Department of Structural Biology Molecular Biology Institute of Barcelona Barcelona Science Park c Baldiri Reixac 15 21 08028 Barcelona Catalonia Spain

Scientific and Technological Centers University of Barcelona Lluís Solé i Sabaris 1 3 08028 Barcelona Catalonia Spain

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