Membrane-Proximal Domain of a Disintegrin and Metalloprotease-17 Represents the Putative Molecular Switch of Its Shedding Activity Operated by Protein-disulfide Isomerase

Abstract

A disintegrin and metalloprotease-17 (ADAM17) is a major sheddase responsible for the regulation of a wide range of biological processes, like cellular differentiation, regeneration, or cancer progression. Hitherto, the mechanism regulating the enzymatic activity of ADAM17 is poorly understood. Recently, protein-disulfide isomerase (PDI) was shown to interact with ADAM17 and to down-regulate its enzymatic activity. Here we demonstrate by NMR spectroscopy and tandem-mass spectrometry that PDI directly interacts with the membrane-proximal domain (MPD), a domain of ADAM17 involved in its dimerization and substrate recognition. PDI catalyzes an isomerization of disulfide bridges within the thioredoxin motif C600XXC603 of the MPD and results in a drastic structural change between an active open state and an inactive closed conformation. This conformational change of the MPD putatively acts as a molecular switch, facilitating a global reorientation of the extracellular domains in ADAM17 and regulating its shedding activity.

Distinct Intracellular Domain Substrate Modifications Selectively Regulate Ectodomain Cleavage of NRG1 or CD44

“…In particular, our own attempts to detect induced dephosphorylation of a tryptic peptide comprising S291 by mass spectrometry have not been successful; this was likely due to the very close proximity of S291 to the membrane, causing reduced detectability of the tryptic peptide in mass spectrometry. However, by 32 P incorporation we were able to show that the CD44 WT and a CD44 mutant with an ICD truncation right after S291 (Fig. 4A, ⌬afterS291) were phosphorylated in vitro (Fig.…”

“…Indeed, ADAMs (A-disintegrin and -metalloproteases), in particular ADAM17, are regulated by several mechanisms that affect their activity, including the level of their expression, trafficking from intracellular compartments to the cell surface (their site of action), removal of the inhibitory prodomain (reviewed in reference 2), and modulation of their catalytic ectodomain structure (30). The last can involve redox regulation targeted to the outside of the cell that induces irreversible changes in the ADAM17 membrane-proximal CANDIS domain relevant for interaction with some select ADAM17 substrates (31)(32)(33). C-terminal phosphorylation of ADAM17 has been reported to increase its surface levels and releases ADAM17 dimers from their inhibitory interaction with the extracellular inhibitor TIMP3 to form presumably active monomers (34,35).…”

A Disintegrin and Metalloprotease 17 Dynamic Interaction Sequence, the Sweet Tooth for the Human Interleukin 6 Receptor

“…After incubation, the beads were washed with PBS (pH 7.4), and the suspension was divided into two parts that were either treated with 10 M reduced PDI in PBS or with PBS only for 15 min at room temperature. The reduced PDI was prepared as described earlier (19). After another washing step with lysis buffer plus 5 mM CaCl 2 , both aliquots of beads were incubated with the human IL-6R lysate for 30 min on ice.…”

“…One isoform is open and elongated, corresponds to the active enzyme, and allows the molecule to be flexible. The second isoform is rigid and compact and corresponds to the inactive form (19). The two isoforms differ in their disulfide bond pattern, and the protein-disulfide isomerase (PDI) controls ADAM17 activity by converting the flexible active MPD into the rigid inactive one (19).…”

“…Thereby, two disulfide bounds undergo a specific isomerization. This process changes the affected disulfide pattern from a sequential arrangement (Cys 600 -Cys 630 and Cys 635 -Cys 641 ), which is present in the elongated structure, to an overlapping pattern (Cys 600 -Cys 635 and Cys 630 -Cys 641 ), which exists in the compact, structured MPD and is associated with the inactive enzyme (19).…”