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

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 multiangle 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.

ADAMTS13 conformations and mechanism of inhibition in immune thrombotic thrombocytopenic purpura

“…1,[38][39][40][41] Recently, cross-linking mass spectrometry was used to demonstrate that, when bound to a surrogate substrate (VWF73), the metalloprotease, disintegrin, cysteine-rich, and spacer domains undergo a significant rearrangement, with the first TSP1 repeat functioning as a "hinge" region, placing the spacer domain, which is the primary determinant of substrate specificity, in close proximity to the metalloprotease domain. 43 This likely occurs via a series of low-affinity interactions with VWF at various exosites that causes dozens, if not hundreds, of small refolding events. The authors describe this as a "fuzzy complex" with a "dynamic zipper" mechanism, offering evidence that ADAMTS13 is likely an intrinsically disordered protein.…”

“…Secreted ADAMTS13 protein consists of a metalloprotease (M) domain, a disintegrin (D) domain, the first thrombospondin type 1 (TSP1‐1) repeat, the Cys‐rich (C) and spacer (S) domains, followed by 7 more TSP1 repeats and two CUB domains, associated with complement components C1r/C1s, sea Urchin epidermal growth factor, Bone morphogenetic protein (Figure 1A). While the tertiary structure of a full‐length ADAMTS13 protein has yet to be determined, the truncated versions of recombinant ADAMTS13 protein (e.g., MDTCS or its variants) have recently been described using a variety of techniques, including X‐ray crystallography, small angle X‐ray scattering, and mass spectrometry plus hydrogen/deuterium exchange, and cross‐linking mass spectrometry 27,28,42,43 . The MDTCS fragment contains all the necessary and sufficient domains of ADAMTS13 protein for substrate specificity and efficient cleavage 28,43,44,45 .…”

“…This poses a significant challenge to structural biologists since ADAMTS13 was first discovered and cloned in 2001 1,38‐41 . Recently, cross‐linking mass spectrometry was used to demonstrate that, when bound to a surrogate substrate (VWF73), the metalloprotease, disintegrin, cysteine‐rich, and spacer domains undergo a significant rearrangement, with the first TSP1 repeat functioning as a “hinge” region, placing the spacer domain, which is the primary determinant of substrate specificity, in close proximity to the metalloprotease domain 43 . This likely occurs via a series of low‐affinity interactions with VWF at various exosites that causes dozens, if not hundreds, of small refolding events.…”

“…This likely occurs via a series of low‐affinity interactions with VWF at various exosites that causes dozens, if not hundreds, of small refolding events. The authors describe this as a “fuzzy complex” with a “dynamic zipper” mechanism, offering evidence that ADAMTS13 is likely an intrinsically disordered protein 43 …”

“…27,28,42,43 The MDTCS fragment contains all the necessary and sufficient domains of ADAMTS13 protein for substrate specificity and efficient cleavage. 28,43,44,45 From these studies, coupled with functional assays, the spacer domain of ADAMTS13 has been identified to be crucial for substrate cleavage and specificity. [45][46][47][48] The crystal structure of the DCTS fragment of ADAMTS13 was reported in 2009, 44 which demonstrates that the spacer domain is highly structured, with 10 beta sheets forming a "sandwich" that exposes various loops.…”