Final Activity Report Summary - NCT-BINDING (Substrate docking and affinity studies of Nicastrin: the binding mechanism.) The gamma-secretase complex is responsible for the proteolysis of integral membrane proteins. Nicastrin (NCT) was proposed to operate as the substrate receptor of the complex with the glutamate 332, Glu(333) in humans, serving as the anionic binding site for the alpha-amino-terminal group of substrates. The putative binding site was located within the aminopeptidase-like domain of NCT. The Glu(332) was proposed to function as the counterpart of the exopeptidase Glu located in the active site of these peptidases. Although Glu(332) could bind the alpha-amino-terminal group of substrates, we hypothesised, in analogy with M28-aminopeptidases, that other residues in the putative binding site of NCT should participate in the interaction as well. Surprisingly, mutagenesis of these residues affected the in vivo processing of amyloid precursor protein (APP) and Notch substrates only weakly. In addition, the E332Q mutation, which completely abolished the anionic alpha-amino-terminal binding function, remained fully active. When we introduced the previously characterised E332A mutation, we found strongly decreased gamma-secretase complex levels; nevertheless the remaining complex appeared as active as the wild-type complex. We confirmed, in two independent in vitro assays, that the specific enzymatic activity of E332A mutant was comparable to that of the wild-type complex. Thus, Glu(332) crucially affected complex maturation rather than substrate recognition. Moreover, other NCT mutants, designed to either impede or alter substantially the putative binding pocket, affected only marginally gamma-secretase activity. Consequently, these studies indicated that the main role of Glu(332) relied in the maturation and assembly of gamma-secretase rather than in the substrates recognition. Even though our experiments did not rule out the fact that the ectodomain of NCT also participated in the enzymatic function of the gamma-secretase complex, our results clearly demonstrated that the previously proposed mechanism could not be maintained as an explanation for the available data. Hence, a discussion was reopened regarding NCT function in the gamma-secretase-mediated regulated intramembrane proteolysis.