A model for the conformational activation of the structurally quiescent metalloprotease ADAMTS13 by von Willebrand factor

Kieron South,Marta O Freitas,David A Lane

doi:10.1074/jbc.m117.776732

Kieron South, Marta O Freitas + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m117.776732

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Abstract

Blood loss is prevented by the multidomain glycoprotein von Willebrand factor (VWF), which binds exposed collagen at damaged vessels and captures platelets. VWF is regulated by the metalloprotease ADAMTS13, which in turn is conformationally activated by VWF. To delineate the structural requirements for VWF-mediated conformational activation of ADAMTS13, we performed binding and functional studies with a panel of truncated ADAMTS13 variants. We demonstrate that both the isolated CUB1 and CUB2 domains in ADAMTS13 bind to the spacer domain exosite of a truncated ADAMTS13 variant, MDTCS (KD of 135 ± 1 0.1 nm and 86.9 ± 9.0 nm, respectively). However, only the CUB1 domain inhibited proteolytic activity of MDTCS. Moreover, ADAMTS13ΔCUB2, unlike ADAMTS13ΔCUB1-2, exhibited activity similar to wild-type ADAMTS13 and could be activated by VWF D4-CK. The CUB2 domain is, therefore, not essential for maintaining the inactive conformation of ADAMTS13. Both CUB domains could bind to the VWF D4-CK domain fragment (KD of 53.7 ± 2.1 nm and 84.3 ± 2.0 nm, respectively). However, deletion of both CUB domains did not prevent VWF D4-CK binding, suggesting that competition for CUB-domain binding to the spacer domain is not the dominant mechanism behind the conformational activation. ADAMTS13ΔTSP8-CUB2 could no longer bind to VWF D4-CK, and deletion of TSP8 abrogated ADAMTS13 conformational activation. These findings support an ADAMTS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.

Highlights

Through its surface-exposed A3 domain [1,2,3]
We first conducted a preliminary co-immunoprecipitation investigation of the binding between the isolated CUB1 and CUB2 domain fragments and the truncated ADAMTS13 variant, MDTCS. Both CUB1 and CUB2 were shown to interact with WT MDTCS in solution with an affinity that was sufficient to allow specific co-immunoprecipitation of the CUB fragments when MDTCS was pulled down
There was an ϳ2-fold increase in the Bmax of binding between WT MDTCS and CUB1-2 compared with the isolated CUB1 and CUB2 domain fragments (Fig. 1G)

Summary

To whom correspondence may be addressed

Through its surface-exposed A3 domain [1,2,3]. Once tethered to collagen, rheological shear forces induce a conformational change [4]. The multimeric size and hemostatic potential of VWF is regulated through a multistep mechanism of proteolysis of its A2 domain by the metalloprotease ADAMTS13 [15, 16] This process begins with a positioning interaction between the C-terminal domains of ADAMTS13 and the C-terminal D4-CK domains of globular VWF [17, 18]. The VWF D4-CK domain region is essential for enhancing activity of ADAMTS13 [31, 32], indicating that the positioning interaction between VWF D4-CK and ADAMTS13 [17] actuates the conformational change required to expose the functionally important spacer domain exosite. Several distinct interactions between VWF D4-CK and ADAMTS13 have been identified, with the TSP8 domain of ADAMTS13 proving essential for VWF D4-CK-induced activation

Results

Discussion

Experimental procedures