Abstract
Introduction: ADAMTS13 is a multidomain protein with metalloprotease (M), disintegrin-like (D), thrombospondin-1 (T), Cys-rich (C) and spacer (S) domains, followed by 7 T domains and 2 CUB domains. ADAMTS13 cleaves the cryptic Tyr1605-Met1606 bond in the A2 domain of von Willebrand factor (VWF), which inhibits the growth of platelet-rich thrombi. When subjected to tensile stress in solution, bound to platelets, or on endothelial cell surfaces, VWF interacts with multiple exosites on ADAMTS13, changing the conformation of both proteins. These close contacts enhance the specific interaction between ADAMTS13 and VWF in vivo. Interactions between VWF and proximal MDTCS domains of ADAMTS13 have been investigated extensively. ADAMTS13 distal domains T8-CUB2 are required to bind the D4 domain of VWF, and the CUB domains promote the cleavage of platelet-decorated VWF strings. Using mutagenesis, small-angle X-ray scattering (SAXS) and enzyme kinetics, we have shown that ADAMTS13 adopts a folded conformation with distal T8-CUB2 domains close to the proximal MDTCS domains and a hinge point between the T4 and T5 domains. We have used internal deletions of T domains to identify a possible "minimal" structure for a functional and stably folded ADAMTS13.
Methods: Recombinant human hADAMTS13, pigeon pADAMTS13, and various T domain deletion mutations were produced in T-Rex 293 cell lines and purified to homogeneity. Similar inactive variants of each protein were prepared with the mutation E225Q, which abolishes catalytic activity but does not affect protein folding. SAXS data were collected at the SIBYLS beamline (Lawrence Berkeley National Laboratory) for ADAMTS13 and deletion variants. The radius of gyration (Rg) and maximum particle size (Dmax) were calculated from scattering profiles using DATGNOM. Ab initio envelopes were generated from scattering profiles using DAMMIN. A molecular model of ADAMTS13 was built from crystal structures of ADAMTS4 MD domains (2rjp) and ADAMTS13 DTCS domains (3ghm), and using HHpred to model distal T domains, CUB domains, and linkers after T4 and T8. ADAMTS13 activity assays were performed at pH 6.0 and pH 7.4 with the fluorogenic substrate FRETS-rVWF71. Monoclonal antibodies (Mabs) against different distal domains of ADAMTS13 and recombinant VWF D4 domain were added to assess allosteric activation.
Results: Rg and Dmax of ADAMTS13 variants are shown in Table 1. For Del4L, with deletion of linker region between T4 and T5, values of Rg and Dmax are ~5% smaller than for full length ADAMTS13. Deletion of both T4 and the linker region (Del4pL), reduced Rg and Dmax ~10%. Deletions of distal domains (Del7, Del8, Del8L, and Del8pL) that are spatially close to the proximal domains increased Rg and Dmax ~14%, which is consistent with partial unfolding of the condensed full length ADAMTS13 structure. Del2to8, which lacks all distal T domains between S and CUBs, aggregated in solution. Del2to7, which retains only T8, has Rg and Dmax 15% and 17% smaller than ADAMTS13, respectively. The kcat values for all variants at pH 7.4 are consistent with autoinhibition of the proximal MDTCS domains by any remaining distal domains. The additional of Mabs recognizing T7, T8 and CUBs slightly (~1.5 fold) activated individual deletion mutants. Del2to7, Del2to8, and Del3to6 had similar activities when compared with ADAMTS13. Though Del2to7 and Del3to6 could be activated by Mabs ~3 fold, only Del3to6 could be activated slightly ~1.3-fold by D4 domain, compared to 1.8-fold activation of hADAMTS13. pADAMTS13 is structurally similar to human Del3to6, and was activated by D4 domain ~4 fold but not by Mabs. Fitting to the ab initio envelopes show a folded conformation for hADAMTS13, Del7, Del8 and Del8L (Figure 1), consistent with a hinge in the flexible linker after T4. pADAMTS13 and Del2to7 had more compact envelopes, consistent with removal of most of the folded segment present in full length hADAMTS13.
Conclusions: Deletion of individual distal T domains does not relieve the autoinhibition of ADAMTS13. Del3to6 is the only human deletion mutant examined that can be activated by both Mabs and VWF D4 domain, similar to hADAMTS13. pADAMTS13 is structurally similar to Del3to6, retains normal activation by VWF D4, and shows a SAXS envelope consistent with a truncated version of hADAMTS13. Our findings suggest that Del3to6 and pADAMTS13 represent the "minimal" structure of allosterically-regulated, functional ADAMTS13.
Sadler:Ablynx: Consultancy; 23andMe: Consultancy; BioMarin: Consultancy.
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