Exploring the "minimal" structure of a functional ADAMTS13 by mutagenesis and small-angle X-ray scattering

Jian Zhu, Joshua Muia, Garima Gupta, Lisa A. Westfield, Karen Vanhoorelbeke, Niraj H. Tolia, J. Evan Sadler

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Human ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C), and spacer (S) domains, followed by 7 additional T domains and 2 CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. ADAMTS13 inhibits the growth of von Willebrand factor (VWF)-platelet aggregates by cleaving the cryptic Tyr1605-Met1606 bond in the VWF A2 domain. ADAMTS13 is regulated by substrate-induced allosteric activation; without shear stress, the distal T8-CUB domains markedly inhibit VWF cleavage, and binding of VWF domain D4 or selected monoclonal antibodies (MAbs) to distal ADAMTS13 domains relieves this autoinhibition. By small angle X-ray scattering (SAXS), ADAMTS13 adopts a hairpin-like conformation with distal T7-CUB domains close to the proximal MDTCS domains and a hinge point between T4 and T5. The hairpin projects like a handle away from the core MDTCS and T7-CUB complex and contains distal T domains that are dispensable for allosteric regulation. Truncated constructs that lack the T8-CUB domains are not autoinhibited and cannot be activated by VWF D4 but retain the hairpin fold. Allosteric activation by VWF D4 requires T7, T8, and the 58-amino acid residue linker between T8 and CUB1. Deletion of T3 to T6 produced the smallest construct (delT3-6) examined that could be activated by MAbs and VWF D4. Columba livia (pigeon) ADAMTS13 (pADAMTS13) resembles human delT3-6, retains normal activation by VWF D4, and has a SAXS envelope consistent with amputation of the hairpin containing the dispensable T domains of human ADAMTS13. Our findings suggest that human delT3-6 and pADAMTS13 approach a "minimal" structure for allosterically regulated ADAMTS13.

Original languageEnglish
Pages (from-to)1909-1918
Number of pages10
JournalBlood
Volume133
Issue number17
StatePublished - 25 Apr 2019
Externally publishedYes

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    Zhu, J., Muia, J., Gupta, G., Westfield, L. A., Vanhoorelbeke, K., Tolia, N. H., & Sadler, J. E. (2019). Exploring the "minimal" structure of a functional ADAMTS13 by mutagenesis and small-angle X-ray scattering. Blood, 133(17), 1909-1918.