Faculty Collaboration Database

Linker regions and flexibility around the metalloprotease domain account for conformational activation of ADAMTS-13. J Thromb Haemost 2015 Nov;13(11):2063-75

Date

09/24/2015

Pubmed ID

26391536

Pubmed Central ID

PMC4778570

DOI

10.1111/jth.13149

Scopus ID

2-s2.0-84946572015 (requires institutional sign-in at Scopus site)   58 Citations

Abstract

BACKGROUND: Recently, conformational activation of ADAMTS-13 was identified. This mechanism showed the evolution from a condensed conformation, in which the proximal MDTCS and distal T2-CUB2 domains are in close contact with each other, to an activated, open structure due to binding with von Willebrand factor (VWF).

OBJECTIVES: Identification of cryptic epitope/exosite exposure after conformational activation and of sites of flexibility in ADAMTS-13.

METHODS: The activating effect of 25 anti-T2-CUB2 antibodies was studied in the FRETS-VWF73 and the vortex assay. Cryptic epitope/exosite exposure was determined with ELISA and VWF binding assay. The molecular basis for flexibility was hypothesized through rapid automatic detection and alignment of repeats (RADAR) analysis, tested with ELISA using deletion variants and visualized using electron microscopy.

RESULTS: Eleven activating anti-ADAMTS-13 antibodies, directed against the T5-CUB2 domains, were identified in the FRETS-VWF73 assay. RADAR analysis identified three linker regions in the distal domains. Interestingly, identification of an antibody recognizing a cryptic epitope in the metalloprotease domain confirmed the contribution of these linker regions to conformational activation of the enzyme. The proof of flexibility around both the T2 and metalloprotease domains, as shown by by electron microscopy, further supported this contribution. In addition, cryptic epitope exposure was identified in the distal domains, because activating anti-T2-CUB2 antibodies increased the binding to folded VWF up to ~3-fold.

CONCLUSION: Conformational activation of ADAMTS-13 leads to cryptic epitope/exosite exposure in both proximal and distal domains, subsequently inducing increased activity. Furthermore, three linker regions in the distal domains are responsible for flexibility and enable the interaction between the proximal and the T8-CUB2 domains.

Author List

Deforche L, Roose E, Vandenbulcke A, Vandeputte N, Feys HB, Springer TA, Mi LZ, Muia J, Sadler JE, Soejima K, Rottensteiner H, Deckmyn H, De Meyer SF, Vanhoorelbeke K

Author

Joshua Muia PhD Assistant Professor in the Biochemistry department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

ADAM Proteins
ADAMTS13 Protein
Allosteric Regulation
Allosteric Site
Amino Acid Sequence
Antibodies, Monoclonal
Antigen-Antibody Reactions
Catalysis
Consensus Sequence
Enzyme Activation
Epitopes
Humans
Microscopy, Electron
Molecular Sequence Data
Protein Binding
Protein Conformation
Protein Folding
Protein Processing, Post-Translational
Protein Structure, Tertiary
Sequence Alignment
Sequence Homology, Amino Acid
Thrombospondin 1
von Willebrand Factor