An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution. Issue 13 (25th June 2021)

• Record Type: Journal Article
• Authors: del Amo-Maestro, Laura; Sagar, Amin; Pompach, Petr; Goulas, Theodoros; Scavenius, Carsten; Ferrero, Diego S.; Castrillo-Briceño, Mariana; Taulés, Marta; Enghild, Jan J.; Bernadó, Pau; Gomis-Rüth, F. Xavier
• Is Part Of: Journal of molecular biology. Volume 433:Issue 13(2021)
• Publication Date: 2021-06-25
• Subjects: protein–protein interactions -- metallopeptidase -- biophysical techniques -- platelet aggregation -- blood coagulation; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2021.166954
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: Highlights: Von Willebrand Factor (vWF) is cleaved at a single site by peptidase ADAMTS-13. We characterised the complex between a vWF-peptide and ADAMTS-13 by nine techniques. The interaction conforms to a fuzzy complex that follows a dynamic zipper mechanism. Many reversible, weak but additive interactions result in strong binding and cleavage. Abstract: 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.