Project Summary. Human insulin-degrading enzyme (IDE) is a highly conserved dimeric zinc metalloprotease that hydrolyzes various peptide substrates, such as insulin, amyloid-β (Aβ), glucagon, amylin, and HIV-1 p6. It is implicated in several physiological and pathological processes, including insulin catabolism, amyloid-β (Aβ) clearance, development of type II diabetes and Alzheimer’s disease (AD), as well as cognitive disorders and glucose intolerance observed among people living with HIV. Surprisingly, in addition to hydrolyzing Aβ, IDE also acts as a nonproteolytic chaperone against Aβ, resulting in its sequestration, followed by its controlled disposal. We recently investigated the interactions of Aβ and HIV-1 p6 with catalytically inactive IDE using relaxation-based solution NMR methods. We uncovered that modulation of intermolecular interactions allows IDE to differentiate between non-amyloidogenic p6 and amyloidogenic Aβ. We also discovered that catalytically inactive IDE prevented Aβ fibrillization at substoichiometric concentrations. The projects in this R21 proposal expand upon these discoveries and will carry out innovative structure-function studies of IDE. Specifically, we will address two outstanding questions in the field of IDE structural biology: the allosteric modulation of its catalytic activity (aim 1) and its remarkable nonproteolytic chaperone activity against Aβ (aim 2). Aim 1 is centered on our hypothesis that substrate-induced closure of one IDE subunit will accelerate the opening of the other, allowing the products to be released or substrate captured, and will provide key insights into how the substrate triggers these conformational transitions as well as the complex network of intrasubunit and intersubunit interactions that govern the catalytic activity of IDE. Under aim 2, we will generate a detailed quantitative picture of how inactive IDE alters Aβ aggregation kinetics using a synergistic combination of fluorescence and NMR spectroscopy and mathematical models based on the framework of microscopic rate laws and chemical kinetics. Extensive preliminary results, including the production of milligram quantities of multiple catalytically active and inactive IDE variants, and excellent NMR spectra of dimeric IDE acquired by introducing sparsely labelled methyl groups in a perdeuterated environment, indicate the high feasibility of successfully completing the proposed studies. Given the importance of IDE in the pathogenesis of AD, our research also has substantial medical relevance and will lay the necessary foundation for an R01 proposal, geared toward bridging the gap between biophysical and clinical studies and developing selective activators and variants of IDE for the treatment of AD.