Abstract
Insulin degrading enzyme (IDE) plays key roles in degrading peptides vital in type two diabetes, Alzheimer's, inflammation, and other human diseases. However, the process through which IDE recognizes peptides that tend to form amyloid fibrils remained unsolved. We used cryoEM to understand both the apo- and insulin-bound dimeric IDE states, revealing that IDE displays a large opening between the homologous ~55 kDa N- and C-terminal halves to allow selective substrate capture based on size and charge complementarity. We also used cryoEM, X-ray crystallography, SAXS, and HDX-MS to elucidate the molecular basis of how amyloidogenic peptides stabilize the disordered IDE catalytic cleft, thereby inducing selective degradation by substrate-assisted catalysis. Furthermore, our insulin-bound IDE structures explain how IDE processively degrades insulin by stochastically cutting either chain without breaking disulfide bonds. Together, our studies provide a mechanism for how IDE selectively degrades amyloidogenic peptides and offers structural insights for developing IDE-based therapies.
Highlights
Insulin degrading enzyme (IDE) is an evolutionarily conserved, M16 family metalloprotease that controls diverse biological functions in model organisms such as mating and cell division in buddingZhang et al eLife 2018;7:e33572
Because IDE in the open conformation has proved recalcitrant to crystallization, we explored the use of cryogenic electron microscopy (cryoEM) to study these structures
To identify Fabs that bind IDE tightly, we screened a phage-display synthetic Fab library constructed using ‘restricted chemical diversity’ where positions randomized within the complementarity determining regions are biased toward amino acids enriched in antibody paratopes (Miller et al, 2012)
Summary
Insulin degrading enzyme (IDE) is an evolutionarily conserved, M16 family metalloprotease that controls diverse biological functions in model organisms such as mating and cell division in budding. Structures of open and insulin-bound state IDE, two key conformations vital for the IDE catalytic cycle, have remained unsolved (Figure 1A and C). The structure of IDE in complex with the fully unfolded insulin prior to the processive cleavage of insulin has remained unsolved Amyloidogenic peptides such as Ab can form highly toxic oligomers/fibrils, leading to many human disorders (Chiti and Dobson, 2006; Eisenberg and Jucker, 2012; Merlini and Bellotti, 2003). The resulting exposed b-strand binds and stabilizes the catalytic cleft within the IDE door subdomain, leading to stochastic cleavage of these peptides This hypothesis could explain how IDE uses substrate-induced stabilization of the IDE catalytic site to selectively degrade amyloidogenic peptides. We integrate all four aforementioned structure methods to elucidate the molecular basis of how IDE captures, unfolds, and degrades its substrates and how IDE recognizes amyloidogenic peptides
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