A novel mechanism underlying allosteric regulation of ADAMTS-13 revealed by hydrogen−deuterium exchange plus mass spectrometry

Background ADAMTS-13, a plasma metalloprotease, cleaves von Willebrand factor. ADAMTS-13 activity appears to be regulated through allosteric inhibition by its distal C-terminus. Objectives The objective of this study was to better understand how domain–domain interactions may affect ADAMTS-13 conformations and functions. Methods We performed deuterium–hydrogen exchange plus mass spectrometry to assess the number and rate of deuterium incorporation into various peptides of full-length ADAMTS-13 and its truncated variants. Results Under physiological conditions, a bimodal distribution of deuterium incorporation was detected in the peptides from metalloprotease (217-230 and 282-304), cysteine-rich (446-482), and CUB (for complement C1r/C1s, Uegf, Bmp1) domains (1185-1214, 1313-1330, 1341-1347, 1358-1378, and 1393-1407) of full-length recombinant ADAMTS-13, but not of truncated variants. These results suggest that the full-length ADAMTS-13 undergoes conformational changes. On removal of the middle and distal C-terminal domains, the number and rate of deuterium incorporation were increased in the peptides from cysteine-rich (445-467, 467-482, and 495-503) and spacer domains (621-642 and 655-654) but decreased in the peptides from metalloprotease (115-124, 217-230, and 274-281). Moreover, most peptides, except for 217-230 and 1357-1376, exhibited a pD-dependent deuterium incorporation in the full-length ADAMTS-13, but not in the truncated variant (eg, MDTCS or T5C). These results further suggest that the bimodal deuterium incorporation observed in the peptides from the full-length ADAMTS-13 is the result of potential impact from the middle to distal C-terminal domains. Surface plasmon resonance revealed the direct binding interactions between the distal and proximal domains of ADAMTS-13. Conclusion Our results provide novel insight on how intramolecular interactions may affect conformations of ADAMTS-13, thus regulating its proteolytic functions.


| I N T R O D U C T I O N
A multidomain and zinc-dependent metalloprotease ADAMTS-13 consists of proximal and distal segments. The proximal segment includes a metalloprotease, a disintegrin-like domain, the first thrombospondin type 1 repeat, a cysteine-rich, and a spacer domain. The distal segment contains 7 more TSP1 repeats and 2 CUB domains (for complement C1r/C1s, Uegf, Bmp1) [1,2]. ADAMTS-13 is synthesized in hepatic stellate cells [3,4], endothelial cells [4][5][6], and perhaps megakaryocytes [7] and is released into the blood stream, where it cleaves newly released ultra large von Willebrand factor (ULVWF) [8,9]. The cleavage of ULVWF by ADAMTS-13 occurs specifically at the Tyr 1605 -Met 1606 bond [10]. This proteolytic cleavage is essential for the regulation of von Willebrand factor (VWF) adhesive function and thrombus formation at the sites of vascular injury.
ADAMTS-13 activity is primarily regulated at its substrate level through fluidic shear [10,26], which causes stretch and conformational changes in the central A2 domain of VWF, allowing ADAMTS-13 to access to the cleavage site. Our previous studies have demonstrated that binding of protein cofactors such as coagulation factor VIII [27], platelet glycoprotein 1b [28,29], and apoB100/LDL [30] may facilitate Essentials • The mechanism underlying allosteric regulation of ADAMTS-13 activity is not fully understood.
• Biophysical study reveals conformational changes of ADAMTS-13 after truncation or pH changes.
• Biophysical study also detects direct binding between distal and proximal ADAMTS-13 domains.
• Our findings provide insights into how interdomain interactions affect ADAMTS-13 conformation.

PILLAI AND ZHENG
shear-induced conformational changes that accelerate the cleavage of VWF by ADAMTS-13. More recently, several studies have also demonstrated that ADAMTS-13 in solution may exhibit various conformations [31,32], and at least, some are sensitive to the changes in environmental pH [32], metal ions (Ca 2+ and Zn 2+ ) [33,34], and the interaction with VWF substrate [32,35] or antibodies in patients with TTP [32,36]. Additionally, a removal of its distal segment consisting of TSP-1 2 to 8 repeats and CUB domains may also result in dramatic conformational changes, thus increasing ADAMTS-13 activity toward the VWF substrates [32]. Moreover, point mutations in the spacer domain may also activate ADAMTS-13 by disrupting the interaction between the proximal and distal domains [36,37]. However, the data available thus far are largely functional and indirect. The exact mechanism underlying how the domain-domain interactions may affect overall conformation of ADAMTS-13 protein is not known.

| HX-MS basic
The HX-MS is a technique used to elucidate protein-protein interaction, dynamics, and conformational changes [42][43][44]. It measures the isotopic mass change associated with the protein amide backbones with its surrounding, which depends on the overall folding and dynamics of the protein. During labeling, proteins are exposed to deuterated buffer for various times to allow deuterium incorporation into their backbone. An aliquot is then quenched by lowering the pH to 2.5 and temperature to nearly zero, followed by a protease digestion. Desalted proteolytic peptides with the help of chilled reverse phase high performance liquid chromatography are then eluted and sprayed onto a mass spectrometer. Analysis of the MS/ MS data reveals the difference in mass due to deuterium uptake.
Other than structural rearrangement, the H-D exchange rate also depends on temperature, pH, and nature of the amino acid. In addition, it depends on the nature of secondary structure, Hbonding, and solvent accessibility. The H-D exchange rate is relatively lower in the obstructed core areas of the protein than that in the exposed areas ( Figure 1A). The protein was allowed to pass through the protease column and was later electro sprayed onto a mass spectrometer as described above ( Figure 1B). Peptides were identified and analyzed for carrying D-label by the ExMS program at peptide resolution [43][44][45][46].

| Peptide coverage of the full-length ADAMTS-13 and its truncated variants by mass spectrometry
To assess the coverage of peptides, protease digestion, liquid chromatography, and MS, as illustrated in Figure 3A   Moreover, at pH >2.5, the rate of hydrogen and deuterium exchange increased logarithmically as the pH/pD increases. Because of this, the rate of exchange at pH6.0 is 10× slower than that at pH7.0 and 100× slower than that at pH8.0. Following the protease digestion, peptides were separated with liquid chromatography and identified by mass     Previous studies have demonstrated that binding of VWF, particularly through its D4 domain [36,60] or a monoclonal antibody against the distal domains of ADAMTS-13 [32,36], to a full-length ADAMTS-13 may induce its conformational changes and increase ADAMTS-13 activity. In addition, lowering pH in the assay buffers (to 6.0) can increase ADAMTS-13 activity by 3-fold [32]. Low pH may disrupt the close contact between the distal and proximal domains of ADAMTS-13, thus removing the allosteric inhibition of ADAMTS-13 by the distal C-terminal domains [32]. This hypothesis is supported by the increase of proteolytic activity and the loss of pH-dependent regulation of the MDTCS, which lacks the distal inhibitory domains [32].
Previous studies also suggest that the integrin recognition  indicate that in the absence of domain-domain interactions between the proximal and distal domains of ADAMTS-13, these peptides are more exposed to the surface and more accessible to the deuterium.
Such a conformational change may explain why MDTCS has more proteolytic activity toward VWF substrates [62].
Moreover, overall folding of the CUB domain is highly dependent on amino acid residues such as L1221, E1267, V1270, G1302, F1304, and G1305 [59]. Similarly, the peptide regions reported in our study contains 2 (eg, E1387 and E1389) of the 6 amino acids responsible for spacer-CUB interaction [59]. In addition, our current result supports their identification of amino acid residues that had an intermediate effect (eg, K1265 and E1382) on spacer-CUB binding [59]. Overall activity of ADAMTS-13 at a given pH depends on its proton change. It is well known about the role of histidines (eg, H224, H228, and H234) and E225 for ADAMTS-13 activity. Optimum substrate binding needs rotational and translational freedom that is greatly influenced by the overall pH changes. In addition, histidine residues in the spacer domain (especially, 556-685) also play a significant role on its pHdependent activity. The pH preference and bimodal changes in this region support the hypothesis in our study. Because we were unable to get a peptide region containing the H1364 in the CUB domain, we could not determine the overall protonation effect of this residue that may be responsible for an enhanced ADAMTS-13 activity at pH6.0 [59].
It remains to be determined where exactly the distal domains interact with the proximal domains. It is possible that the distal CUB

FUNDING
The study is supported by grants from National Heart Lung and Blood Institute (HL126724, HL144552, and HL157975 to X.L.Z.).

AUTHOR CONTRIBUTIONS
V.G.P. and X.L.Z. designed research, performed experiments, and analyzed the results, as well as wrote the manuscript. All authors read and approved the final paper.
is also the Co-Founder of Clotsolution, Inc. V.G.P has declared no relevant conflict.

DATA AVAILABILITY
The raw data sets for the hydrogen−deuterium exchange plus MS experiments are deposited in the public accessible Massive Data Repository at the Center for Computational MS, the University of F I G U R E 7 Deuterium incorporation in the peptides from the full-length ADAMTS13 and the truncated variants at pD6.0 over time in comparison to that of the same peptides at pD8.0. The deuterium incorporation rates in the peptides derived from the N-terminal portion the full-length ADAMTS13 (A) and the MDTCS fragment (B) at pD6.0 compared with that at pD8.0 at 2 different time points. In addition, the deuterium incorporation rates of the peptides derived from C-terminus of a full-length ADAMTS13 (C) and T5C (D) at pD6.0 compared with that of the same peptides at pD8.0 at 2 different time points. The deuterium incorporation rate is approximately 100× slower at pD6.0 than at pD8.0. Thus, the comparison of the deuterium incorporation was made between 6000 seconds at pD6.0 and 60 seconds at pD8.0, for instance. Here, Met, Dis, CysR, Spa, CUB1, and CUB2 indicate metalloprotease, disintegrin, cysteine-rich, spacer, CUB-1 and CUB-2 domains, respectively. The purple or blue colors indicate an increase of deuterium incorporation rate at pD6.0 compared with pD8.0. Yellow indicates the peptides with same deuterium incorporation rate at pD6.0 and pD8.0. Gray shows the areas of no peptides recovered.

PILLAI AND ZHENG
California, San Diego. The specific ID for deposited data is MSV000087189.