Molecular Basis of Catalytic Chamber-assisted Unfolding and Cleavage of Human Insulin by Human Insulin-degrading Enzyme

Abstract

Insulin is a hormone vital for glucose homeostasis, and insulindegrading enzyme (IDE) plays a key role in its clearance. IDE exhibits a remarkable specificity to degrade insulin without breaking the disulfide bonds that hold the insulin A and B chains together. Using Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to obtain high mass accuracy, and electron capture dissociation (ECD) to selectively break the disulfide bonds in gas phase fragmentation, we determined the cleavage sites and composition of human insulin fragments generated by human IDE. Our timedependent analysis of IDE-digested insulin fragments reveals that IDE is highly processive in its initial cleavage at the middle of both the insulin A and B chains. This ensures that IDE effectively splits insulin into inactive N-and C-terminal halves without breaking the disulfide bonds. To understand the molecular basis of the recognition and unfolding of insulin by IDE, we determined a 2.6-Å resolution insulin-bound IDE structure. Our structure reveals that IDE forms an enclosed catalytic chamber that completely engulfs and intimately interacts with a partially unfolded insulin molecule. This structure also highlights how the unique size, shape, charge distribution, and exosite of the IDE catalytic chamber contribute to its high affinity (ϳ100 nM) for insulin. In addition, this structure shows how IDE utilizes the interaction of its exosite with the N terminus of the insulin A chain as well as other properties of the catalytic chamber to guide the unfolding of insulin and allowing for the processive cleavages. IDE3 is an ϳ110-kDa zinc metalloprotease that is evolutionarily conserved from bacteria to humans (1, 2). It was first discovered based on its high affinity to bind insulin (ϳ100 nM) and degrade it into pieces (3, 4). Insulin is a 5.8-kDa hormone that plays a central role in glucose homeostasis and the development of diabetes in humans. Consistent with the in vitro activity of IDE for insulin degradation, loss-of-function mutations of IDE in rodents result in elevated insulin levels and glucose intolerance (5). In addition, a nucleotide polymorphism of the human IDE gene is linked to type 2 diabetes (6). Later studies showed that IDE can also degrade amyloid-␤ (A␤), a peptide vital to the progression of Alzheimer disease (7,8). Accumulating evidence from rodent models and human genetic analyses also indicate the physiological role of IDE in the clearance of A␤ (5, 9 -12).Despite nearly 60 years of studies on IDE, the molecular basis by which IDE binds, unfolds, and degrades insulin has only begun to be elucidated. Different from ATP-dependent proteases, IDE does not require the additional energy source such as ATP to unfold, bind, and cleave its substrates (4, 13). Insulin consists of the A and B chains that are held together by two inter-and one intra-chain disulfide bonds. Remarkably, IDE does not require disulfide bond isomerase activity to unfold and cleave insulin (4). Thus, IDE needs to overcome the stability created by the d...

Metal ions affect insulin-degrading enzyme activity

“…From these data it comes out that the degradation of B20-30 by IDE is much slower than what was reported for insulin [52], at the peptide/enzyme concentration ratios used. Both the shorter chain length of this peptide [69] and the fact that in full length insulin the cleavages at the end of the B chain occur only after the initial cleavages in the middle of insulin A and B chains [23] might be responsible for the observed slower degradation rate by IDE. On the other hand, catalytic parameters for the cleavage of the B20-30 peptide are quite similar to those observed for the enzymatic processing of Aβ (which showed a three-fold slower k cat and a three-fold higher affinity for substrate, see Ref.…”

“…The complex between IDE and the 1-18 portion of the B chain of insulin (PDB code 2WBY) [23] was used for the alignment of B20-30 prior to docking to IDE. The mutation E111Q present in 2WBY was replaced with E111.…”

“…We carried out enzymatic digestions of B20-30, in the presence or absence of copper(II), aluminum(III) and zinc(II), metals chosen for their important role in neurodegenerative diseases [24,26,66,67]. Moreover, since copper enters the cell as copper(I) through high-affinity plasma membrane copper transporters or low affinity permeases [68] and IDE is mainly cytosolic [1], it is interesting to analyze the effect of both oxidation states on B (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) processing. Furthermore, as silver belongs to the same group of copper and silver(I) has the same chemistry of copper(I), we have also performed studies using this ion, in order to have a replication of the copper(I) results without any possible invalidation due to copper(I) oxidation problems (although we worked in a reducing environment, copper(I) could be oxidized to copper(II) during the incubation with the enzyme).…”

“…To assess how metal ions affect the IDE enzymatic activity, steady state kinetics on the B (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) peptide in the presence and in the absence of the above mentioned ions were performed. Fig.…”

“…Furthermore, several evidences indicate that the selective binding of IDE's substrates and their unfolding and processing are driven by the unique size, shape and electrostatic potential of the catalytic chamber and by interaction with the exosite [22,23]. However the molecular basis of the enzymatic activity modulation has only begun to be elucidated.…”