Wrapping it Up: Structural Basis of ADAMTS13 Global Latency

ADAMTS13 is a critical enzyme responsible for cleaving ultra-large von Willebrand factor (VWF) multimers, thereby preventing the formation of microthrombi in the microvasculature. Dysfunction or deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura (TTP), a rare and life-threatening disorder characterized by microangiopathic hemolytic anemia and severe thrombocytopenia. Understanding the regulation of ADAMTS13 is essential for developing therapeutic interventions for TTP. ADAMTS13 exhibits two latency mechanisms: local latency within the metalloprotease (MP) domain and global latency that affects its overall conformation. Although the role of the two CUB domains in binding the Spacer module is known during global latency, the contributions of TSP7, TSP8, and the flexible Linker region (L3) between TSP8 and CUB1 have been repeatedly observed but never explained. Binding studies with monoclonal antibodies (mAbs) targeting the MP domain have revealed a cryptic epitope that becomes accessible only when ADAMTS13 is conformationally activated, and Spacer-CUB binding is disrupted. However, the mechanism behind this long-range effect has never been explained. We present a novel autoinhibition model of ADAMTS13, proposing that its distal domains directly occlude substrate binding sites, thereby modulating the enzymes activity. In this model, TSP7 and TSP8 bind directly to the MP module, while the Linker region between TSP8 and CUB1 acts as a pseudosubstrate, mimicking the natural VWF-A2 substrate and blocking the binding sites. Our results show that the CUB domains must depart from the tandem arrangement observed in the crystal structure and instead adopt refined binding positions. Further evidence comes from novel monoclonal antibodies targeting more cryptic epitopes on various ADAMTS13 domains that we have recently presented, as well as pH- and EDTA-dependent conformational changes observed by ELISA. A detailed molecular model is derived from extensive molecular simulations and mechanistically reconciles a wide range of previously disconnected experimental observations. This novel insight into the regulation of ADAMTS13 lays the groundwork for new therapeutic strategies against TTP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/689657v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@abed60org.highwire.dtl.DTLVardef@16d7470org.highwire.dtl.DTLVardef@f7a14aorg.highwire.dtl.DTLVardef@17e350f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIAtomistic Structure of autoinhibited ADAMTS13-Del3To6 C_LIO_LIL3 linker (TSP8-CUB1) has a dual function: acting as a pseudosubstrate that blocks VWF-A2 binding sites and guiding CUB1-2 positioning C_LIO_LITSP7 and TSP8 bind at the MP domain to restrain activity C_LIO_LICUB1 and CUB2 domains are not a single functional block C_LI