Ensemble cryoEM elucidates the mechanism of insulin capture and degradation by human insulin degrading enzyme

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.

Reassessment of an Innovative Insulin Analogue Excludes Protracted Action yet Highlights the Distinction between External and Internal Diselenide Bridges

“…Although whether IDE is indeed a valid target for diabetes‐specific drug design is uncertain, such an analogue could provide an elegant reagent with which to test the physiologic hypothesis that IDE regulates the duration of insulin action‐a key issue at the frontier of molecular endocrinology. The structural complexity of IDE's internal catalytic chambers highlights the subtlety of this challenge in protein design.…”

Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition

“…Our structural analysis of two members of the cryptidase family, IDE and PreP suggests a common framework for amyloidogenic peptide recognition with distinct specializations that support e cient cytotoxic peptide clearance in their distinct cellular niches 6,22,36,47,48 . Both enzymes belong to the M16 clan of metalloproteases and have homologous ~ 55 kDa N-and C-terminal domains (Fig.…”

“…Furthermore, their catalytic cleft of both enzymes is formed between their N-and C-terminal domains, which is unstable in the absence of substrate. They use substrate-assisted catalysis mechanism to selectively recognize and degrade amyloid peptides (this work) 36,47 . However, there are noticeable differences between IDE and PreP.…”

“…13, Table 2). SAXS is a highly effective technique to eliminate structural models that do not produce calculated scattering patterns that t the experimental scattering pro le 36 . SEC-SAXS also reduces large aggregates that contaminated our previously reported SAXS pro le of PreP 6 .…”

“…Our structural analysis predicts the conformational dynamics of switch A and C and substrate-induced stabilization of three switch regions. To test this, we utilized peptide amide hydrogen deuterium exchange (HDX) mass spectrometry, which is a powerful tool to probe protein conformational dynamics because it allows evaluation of comparative solvent accessibility throughout the protein [36][37][38][39][40][41] . As expected, a reduced HDX was observed in segments in both PreP-N and PreP-C that are involved in the substrate binding (e.g., aa 91-141, aa 703-720, aa 892-903, and aa 931-940).…”