Molecular basis for the recognition and cleavages of IGF-II, TGF-alpha, and amylin by human insulin-degrading enzyme

Abstract

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-beta, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-alpha (TGF-alpha) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-alpha, amylin, reduced amylin, and amyloid-beta by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-alpha at 2.3 A and IDE-amylin at 2.9 A. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.

Figure 1. Characterization of the degradation of IGF-II and TGF-α by IDE

(A) Primary sequences of human IGF-II, TGF-α and insulin. Disulfide bonds are shown by a line connecting two cysteines. Arrows on IGF-II and TGF-α depict the cleavage sites by IDE from this analysis while the cleavage sites of insulin are as reported. The underlined amino acids are observed in the crystal structures of substrate-bound IDE. (B) Comparison of secondary structure (left) and electrostatic surface representation (right) of IGF-II, TGF-α, and insulin. PDB accession codes of IGF-II, TGF-α, and insulin are 1IGL, 1YUF, and 1G7A, respectively. The molecular surface is colored as calculated by APBS (<−6 kT in red, 0 kT in white, and > +6 kT in blue). (C) Inhibition of the IDE-mediated degradation of the fluorogenic substrate V (a bradykinin-mimetic) by insulin, TGF-α and IGF-II. IDE (1 μg) was mixed with substrate V (450 nM) in the presence of indicated concentrations of insulin, TGF-α, and IGF-II and fluorescence intensity was monitored for 10 minutes at 37°C. Results (means ± S.D.) are representative of three independent experiments performed in duplicate. (D) and (E) show the representative MS/MS spectrum for N-terminal regions of IGF-II and TGF-α, respectively. The ESI-tandem mass spectrum of the 529.229 ion from IDE-digested TGF-α atm/z 1584.668 (left) and the 976.469 ion from IDE-digested IGF-II atm/z 1950.936 (right); The amino acid sequence and ion identification of the tandem mass spectrum is shown and experimentally observed ions are labeled in the sequence.

Figure 2. Structures of IDE in complex with amylin

(A) Global view of the structure of amylin-bound IDECF-E111Q monomer. IDE-N and IDE-C are colored green and cyan, respectively. Amylin is in the stick representation colored in orange. (B) Stereo view of composite omit map (purple) of the IDE-bound amylin is contoured at 1.5σ. Oxygen, nitrogen, and carbon atoms of amylin are shown in red, blue, and orange, respectively. (C) Primary sequences of amylin, reduced amylin and Aβ (1-40). The arrows on the amylin depict the cleavage sites by IDE as experimented and the arrows on Aβ (1-40) depict the cleavage sites by IDE after short (1-5 second) incubation. The minor cleavage sites on Aβ(1-40) are marked with the shorter arrows. The underlined amino acids with the scissor bond marked by a red arrow are observed in the crystal structures of substrate-bound IDE. (D) Detailed interaction of the N-terminus of non-reduced amylin (left), reduced amylin (middle) and Aβ(1-42) (right) with the exosite of IDE. (E) Detailed interaction of amylin with the IDE catalytic site. The color of IDE residues corresponds to the respective color of IDE domain in figure 2A. The PDB codes for structures of amylin-bound IDE-CF-E111Q, reduced amylin-bound IDE-E111Q, and Aβ(1-42)-bound IDE-CF-E111Q are 3HGZ, 2G48, and 2WK3, respectively.

Figure 3. Structures of IDE in complex with IGF-II and TGF-α

(A) Global view of the structure of IGF-II bound (top) and TGF-α bound (bottom) IDE-CF-E111Q monomer. IDE-N and IDE-C are colored green and cyan as Figure 1. IGF-II and TGF-α are colored orange. (B) Composite omit maps (blue) of IGF-II and TGF-α are contoured at 1.5σ. The substrates are colored orange. (C) The detailed interaction of the N-terminus of IGF-II and TGF-α with the exosite site of IDE. (D) Detail interaction of IDE catalytic chamber with IGF-II and TGF-α. Atoms oxygen, nitrogen, and carbon of substrates are shown in red, blue, and orange, respectively.

Figure 4

Analysis of IDE's binding with IGF-II, TGF-α, and amylin. (A) A model of how IDE binds, unfolds, and degrades its substrate. IDE has two conformational states: the open state, IDE^o and the closed state IDE^c. The IDE-N and IDE-C are depicted as green and cyan, respectively. IGF-II (PDB code 1IGL) is depicted as red cartoon. IDE^o is theoretically modeled based on the substrate free E. coli pitrilysin structure (1Q21), IDE^c corresponds to the atomic coordinate of IDE-CF-E111Q-IGFII (3E4Z). The detailed description of the mechanism is in the discussion. (B) Shape and surface charge distribution of IGF-II (left), TGF-α (middle) and amylin (right) modeled in the catalytic chamber of IDE. (C) Comparison of IGF-II (left), TGF-α (middle) and amylin (right) in their free forms (transparent grey) with IDE-bound forms (red). The segments in the free forms of IGF-II and TGF-α corresponding to IDE-bound forms are colored in transparent red. The arrows indicate the cleavage sites and the disulfide bonds are colored in yellow. (D) Comparison of IDE-bounded IGF-II (red), TGF-α (green), amylin (blue) and Aβ(1-42) (orange) in stick model.

Figure 5

Structural comparison of IGF, EGF, and amyloidogenic peptides. (A) Sequence alignment of the IGF-II/IGF-I and TGF-α/EGF. (B) Comparison of the electrostatic surface of IGF-II (1IGL), IGF-I (3GF1), TGF-α (1YUF), EGF (1EPH), amylin (2KB8) and Aβ (1-40) (1AML). The molecular surface is colored as calculated by APBS (<−6 kT in red, 0 kT in white, and > +6 kT in blue). The dipole moment of these peptides was the average from distinct NMR solution structures (10 for IGF-I, 20 for IGF-II, 16 for TGF-α, 10 for EGF, 20 for Aβ(1-40), and 30 for amylin). (C) NMR solution structures of IGF-II, IGF-I, TGF-α, EGF, amylin and Aβ (1-40).