Structure of Substrate-free Human Insulin-degrading Enzyme (IDE) and Biophysical Analysis of ATP-induced Conformational Switch of IDE

Abstract

Insulin-degrading enzyme (IDE) is a zinc metalloprotease that hydrolyzes amyloid-␤ (A␤) and insulin, which are peptides associated with Alzheimer disease (AD) and diabetes, respectively. Our previous structural analysis of substratebound human 113-kDa IDE reveals that the N-and C-terminal domains of IDE, IDE-N and IDE-C, make substantial contact to form an enclosed catalytic chamber to entrap its substrates. Furthermore, IDE undergoes a switch between the closed and open conformations for catalysis. Here we report a substrate-free IDE structure in its closed conformation, revealing the molecular details of the active conformation of the catalytic site of IDE and new insights as to how the closed conformation of IDE may be kept in its resting, inactive conformation. We also show that A␤ is degraded more efficiently by IDE carrying destabilizing mutations at the interface of IDE-N and IDE-C (D426C and K899C), resulting in an increase in V max with only minimal changes to K m . Because ATP is known to activate the ability of IDE to degrade short peptides, we investigated the interaction between ATP and activating mutations. We found that these mutations rendered IDE less sensitive to ATP activation, suggesting that ATP might facilitate the transition from the closed state to the open conformation. Consistent with this notion, we found that ATP induced an increase in hydrodynamic radius, a shift in electrophoretic mobility, and changes in secondary structure. Together, our results highlight the importance of the closed conformation for regulating the activity of IDE and provide new molecular details that will facilitate the development of activators and inhibitors of IDE.

Nitric oxide inhibits insulin-degrading enzyme activity and function through S-nitrosylation

“…However, it has also been reported that ATP can increase degradation of small peptides by IDE, while having little effect on insulin metabolism [16,17]. Whatever the effect on insulin degradation, ATP does induce a conformational change in IDE [16]. We have shown that IDE activity is inhibited non-competitively by select free fatty acids and their coenzyme A thioesters [18].…”

“…Camberos et al showed ATP inhibited insulin degradation [15]. However, it has also been reported that ATP can increase degradation of small peptides by IDE, while having little effect on insulin metabolism [16,17]. Whatever the effect on insulin degradation, ATP does induce a conformational change in IDE [16].…”

Dual Exosite-binding Inhibitors of Insulin-degrading Enzyme Challenge Its Role as the Primary Mediator of Insulin Clearance in Vivo

“…The time course of insulin digestion by the dissociating the IDE⅐NTE-2 complex can be obtained by substituting K cat (app) from Equation 7 for the K cat in Equation 5. Fitting of the data were performed using Mathematica software (Wolfram Research).…”

“…Addi-* All authors were employees and shareholders of Eli Lilly and Company at the time this work was completed. tionally, the enzyme is known to be activated by ATP, which destabilizes the closed form of the enzyme (5). Both Türkay and co-workers (17) and Leissring and co-workers (18) have reported the identification of additional unique small molecules that show the ability to activate the enzyme with substrate specificity.…”

“…Of its many substrates, IDE is exceptionally effective at degrading insulin (K m ϭ 85 nM and k cat /K m ϭ 2.42 min Ϫ1 M Ϫ1 ) (4). The IDE active site arises from a clamshell-like structure of the enzyme that consists of two concave halves connected by a linker (5). This creates an overall structure similar to a hollow sphere where the catalytic zinc atom is contained in the cavity ( Fig.…”