Expression and protein chemistry yielding crystallization of the catalytic domain of ADAM17 complexed with a hydroxamate inhibitor.
The membrane-anchored metalloproteinase ADAM17 (TNF-alpha converting enzyme; TACE; EC 3.4.24.86) continues to be an attractive drug target in inflammatory diseases and cancer. Cocrystallization of its catalytic domain with a lead compound was complicated by the tenacious retention of the prodomain that has been shown to be enhanced if ADAM17 is expressed without the disintegrin/cysteine-rich domain that normally follows the N-terminal metalloproteinase. When a truncated form of ADAM17 composed of the signal peptide with the pro- and catalytic domains was expressed in baculovirus-infected insect cells, the major secreted product was a ternary complex of two prodomain fragments with the catalytic domain. The component polypeptides of the ternary complex were characterized by N-terminal analysis and mass spectrometry. Internal cleavage of the propeptide occurred following Arg-58, and a carboxypeptidase variably removed up to three basic residues from the newly created C-terminus. Cleavage at the C-terminus of the propeptide occurred after Arg-214. To prepare ADAM17 for crystal growth, a drug-like inhibitor was used to displace the propeptide and the complex of the catalytic domain with the inhibitor was isolated by size-exclusion chromatography and crystallized.
Hoth
Lise R
LR
Pfizer Global Research and Development, Groton, CT 06340, USA.
Tan
Douglas H
DH
Wang
Ing-Kae
IK
Wengender
Paul A
PA
Thompson
Michelle A
MA
Kamath
Ajith V
AV
Geoghegan
Kieran F
KF
D051722:ADAM Proteins / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000072198:ADAM17 Protein; D020134:Catalytic Domain; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003460:Crystallization; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D013058:Mass Spectrometry
D004791:Enzyme Inhibitors; D006877:Hydroxamic Acids; D051722:ADAM Proteins; D000072198:ADAM17 Protein; C000606462:ADAM17 protein, human