title: Activity of ADAM17 (a disintegrin and metalloprotease 17) is regulated by its noncatalytic domains and secondary structure of its substrates.
abstract: ADAM proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off target toxicity. Targeting secondary substrate binding sites (exosites) can potentially work as an alternative strategy for drug discovery; however, there are only a few reports of potential exosites in ADAM protease structures. In the study presented here, we utilized a series of TNFα-based substrates to probe ADAM10 and 17 interactions with its canonical substrate to identify the structural features that determine ADAM protease substrate specificity. We found that noncatalytic domains of ADAM17 did not directly bind the substrates used in the study but affected the binding nevertheless, most likely because of steric hindrance. Additionally, noncatalytic domains of ADAM17 affected the size/shape of the carbohydrate-binding pocket contained within the catalytic domain of ADAM17. This suggests that noncatalytic domains of ADAM17 play a role in substrate specificity and might help explain differences in substrate repertoires of ADAM17 and its closest homologue, ADAM10. We also addressed the question of which substrate features can affect ADAM protease specificity. We found that all ADAM proteases tested (i.e., ADAM10, 12, and 17) significantly decreased activity when the TNFα-derived sequence was induced into α-helical conformation, suggesting that conformation plays a role in determining ADAM protease substrate specificity. These findings can help in the discovery of ADAM isoform- and substrate-specific inhibitors.
authors.affiliation: Torrey Pines Institute for Molecular Studies, Port St. Lucie, Florida 34987, USA.
authors.forename: Roma
authors.initials: R
authors.lastname: Stawikowska
authors.forename: Mare
authors.initials: M
authors.lastname: Cudic
authors.forename: Marc
authors.initials: M
authors.lastname: Giulianotti
authors.forename: Richard A
authors.initials: RA
authors.lastname: Houghten
authors.forename: Gregg B
authors.initials: GB
authors.lastname: Fields
authors.forename: Dmitriy
authors.initials: D
authors.lastname: Minond
mesh_terms: D051722:ADAM Proteins / Q000737:chemistry / Q000378:metabolism*; D000072198:ADAM17 Protein; D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002942:Circular Dichroism; D006801:Humans; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D013379:Substrate Specificity
chemical_list: D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human
keywords: ADAM ADAMTS; Cell Surface Enzymes; Enzyme Inhibitors; Enzyme Kinetics; Enzyme Mechanisms; Exosite Inhibitor; Exosite Substrate; Noncatalytic Domains; Substrate Conformation