Inhibition of the TNFa Converting Enzyme (TACE) by its Pro Domain

TACE is a disintegrin metalloproteinase that processes tumor necrosis factor and a host of other ectodomains. TACE is biosynthesized as a zymogen and activation requires the removal of an inhibitory pro domain. Little is known about how the pro domain exerts inhibition for this class of enzymes. In order to study the inhibitory properties of the pro domain of TACE, we have expressed it in isolation from the rest of the protease. Here we show that TACE Pro is a stably folded protein that is able to inhibit this enzyme. TACE Pro inhibited the catalytic domain of TACE with an IC50 of 70nM. In contrast, this inhibitory potency decreased over 30-fold against a TACE form containing the catalytic plus disintegrin/cysteine-rich domains (IC50 greater that 2μM). The disintegrin/cysteine rich region in isolation also decreases the interaction of TACE Pro with the catalytic domain. Surprisingly, we found that the cysteine-switch motif located in TACE Pro was not essential for inhibition of TACE’s enzymatic activity: the pro domain variant C184A showed the same inhibitory potency against both TACE forms as wild type TACE Pro. X-ray absorption spectroscopy (XAS) experiments indicate that binding of TACE Pro to the catalytic domain does include ligation of the catalytic zinc ion via the sulfur atom of its conserved Cys184 residue. Moreover, the binding of TACE Pro to the catalytic zinc ion partially oxidizes the catalytic zinc ion of the enzyme. In spite of this, the nature of the interaction between the pro and catalytic domains of TACE is not consistent with a simple competitive model of inhibition based on cysteineswitch ligation of the zinc ion within TACE’s active site. by gest on O cber 1, 2017 hp://w w w .jb.org/ D ow nladed from