Abstract
Background: Deletion of the distal C-terminal domains, or a decrease in pH, or the binding of a monoclonal antibody to the C-terminal domains of ADAMTS13 increases its proteolytic activity towards VWF peptide substrate, VWF73, by approximately 2-fold; this suggests an allosteric interaction between the distal and proximal domains of ADAMTS13. However, neither the underlying mechanism of the allosteric interaction nor the exact sites of interaction between the proximal domains and the distal C-terminal domain are fully understood.
Objective: To determine the binding sites and kinetic interactions between the proximal and distal domains of ADAMTS13 and to gain insight into the mechanism of allosteric regulation of ADAMTS13 function.
Methods: Hydrogen deuterium exchange plus LC-mass spectrometry (HX-MS) and surface plasmon resonance (SPR) were employed to determine conformational changes and direct binding interactions between the proximal and distal domains of ADAMTS13.
Results: Recombinant full-length (rFL-A13) and an ADAMTS13 variant truncated after the spacer domain (rMDTCS) were purified to homogeneity. After incubation with deuterium for various times (0-6 hours), both rFL-A13 and rMDTCS were digested with pepsin and fungal protease-13 and then partially resolved by liquid chromatography before being injected into a LTQ ObitrapXL mass spectrometer for peptide identification. Overall, 188 of the total 338 peptides in rFL-A13 were identified, spanning the metalloprotease, cysteine-rich, and spacer domain. Only 16 peptides were found to be less accessible (or protected from deuterium-hydrogen exchange) in rFL-A13 than in rMDTCS. Two of these regions reside in the metalloprotease domain (residue 78-127 and 282-304). The next region is at a loop on the cysteine-rich domain (residue 446-473 and 495-501). The next peptides are in the adjacent loops of the spacer domain with one peptide 596-603, and five overlapping peptides spanning from 632-642, and then finally one peptide from the C-terminal of the MDTCS variant (residue 676 to 688). These results indicate that the distal C-terminal domains interact with multiple discrete sites in the proximal domains of ADAMTS13, resulting in protection from hydrogen-deuterium exchange. Such an interaction was further demonstrated by direct binding experiments with SPR. The distal domains including CUB, T2-CUB, and T5-CUB bound directly to M, MDT, and MDTCS under flow with high affinity.
Conclusion: Our findings demonstrate the allosteric interactions between the distal domains and the proximal domains at multiple discrete sites. Such an interaction may regulate catalytic efficiency and substrate specificity of the metalloprotease domain of ADAMTS13 under various conditions.
Disclosures
Zheng: Ablynx: Consultancy; Alexion: Research Funding.
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