In regulated intramembrane proteolysis (RIP), an integral membrane protein is cleaved by intramembrane cleaving proteases (iCLiPs) within the transmembrane domain (TM) to liberate biologically active fragments. g- secretase, an aspartyl iCLiP, cleaves within the transmembrane domain of APP (APPTM) to release amyloid-b peptide, which aggregates to form senile plaque in the brain, a pathological hallmark of AD. The long-term goal is to study the g-secretase and APPTM complex and to delineate the atomistic mechanism of g-secretase and APPTM interaction, and the structural mechanism of RIP. Here we will test a novel hypothesis for substrate/enzyme interaction in RIP and we will use APPTM and an archael presenilin homolog (PSH) as a simplified model system for g-secretase. Using solution NMR, mutagenesis and enzyme assays, we will map the binding sites on both the substrate APPTM (aim 1) and the enzyme PSH (aim 2), and finally derive a structural model of substrate docking to the enzyme (aim 3).