Atomic structures of IAPP (amylin) fusions suggest a mechanism for fibrillation and the role of insulin in the process

Abstract

Islet Amyloid Polypeptide (IAPP or amylin) is a peptide hormone produced and stored in the beta-islet cells of the pancreas along with insulin. IAPP readily forms amyloid fibrils in vitro, and the deposition of fibrillar IAPP has been correlated with the pathology of type II diabetes. The mechanism of the conversion that IAPP undergoes from soluble to fibrillar forms has been unclear. By chaperoning IAPP through fusion to maltose binding protein, we find that IAPP can adopt a alpha-helical structure at residues 8-18 and 22-27 and that molecules of IAPP dimerize. Mutational analysis suggests that this dimerization is on the pathway to fibrillation. The structure suggests how IAPP may heterodimerize with insulin, which we confirmed by protein crosslinking. Taken together, these experiments suggest the helical dimerization of IAPP accelerates fibril formation and that insulin impedes fibrillation by blocking the IAPP dimerization interface.

Figure 1

The crystal structure of the maltose binding protein (MBP)-IAPP fusions protein reveals the helical propensity of IAPP. (A) A helix-helix homodimerization interface (green arrow between IAPP molecules) is observed between fusion molecules. The two MBP molecules are shown in blue and yellow, respectively. Residues of the fusion attributable to IAPP are shown in red, each attached to MBP molecules. (B) Space filling representation showing the tight interface between parallel IAPP helices, centered at Phe 15. The view is in the direction of the black arrow in A. (C) By rotating the IAPP homodimer, it can be seen that the 8–18 helices interact at a 55° angle. (D and E) A MBP-IAPP1-22 fusion reveals the cyclic disulfide bond between Cys 2 and Cys 7 and the helical propensity within the N-terminus. MBP is shown in light blue and IAPP is shown in red. (F) View looking down the N-terminal helix of IAPP shows the 3₁₀ helix between residues Gln10 and Val17.

Figure 2

The homodimerization of IAPP suggests an analogous structure for IAPP-insulin heterodimerization. (A) Sequence alignment of residues 8–23 from IAPP with residues 9–24 of the insulin B chain. (B) The putative IAPP-interacting segment from insulin (helical segment composed of residues 9–20 shown in gray) is shown overlaid on the IAPP 8–19 helix. This interaction is centered on the stacked aromatic sidechains (Phe 15 from IAPP and Tyr 16 from insulin) between the two helices. (C) Computationally energy-minimized packing between insulin 9–19 and IAPP 8–18 helices. Notice that the energy-minimized helices are more parallel than the helices in B. (D) Crosslinking of IAPP and Insulin reveals an IAPP dimer (red arrow), an insulin dimer (blue arrow) as well as an IAPP-insulin heterodimer (white arrow). IAPP also crosslinks to higher-order insulin multimers. Myoglobin is included as a negative control for nonspecific crosslinking with IAPP.

Figure 3

Computational docking experiments yielded an ensemble of solutions that suggest how IAPP might dimerize and the effect on the interaction of residue replacements at Phe15. In (A), a docked dimer between wild-type IAPP segments 8–19 reveals a relative rotation of the helices and tighter association than observed in the crystal structure of IAPP. This association disrupts the aromatic stacking between Phe15 residues in exchange for more contact area between the molecules. This association led us to believe that small residue substitutions for Phe15, such as alanine or serine may allow the helices to pack even closer and bury more surface area, as shown in (B) for F15S. The tighter association places Phe15 in closer proximity to His18 and Arg11 of the adjacent molecule, suggesting that acidic residues could potentially stabilize the interaction while large, positively charged residues, such as lysine or arginine could disrupt helical packing. In (C), the F15D dimer reveals how the negative charge may help stabilize the dimer by interacting with Arg11 in the neighboring molecule. In (D), a top scoring interaction model of the IAPP F15D mutant and insulin suggests an alternative interaction mode between IAPP and insulin as compared to that in Figure 2. The rotated IAPP helix exposes Asp15 to solvent and buries several hydrophobic residues from both IAPP and insulin. This packing was not observed in the MBP-fusion structure presumably because the Phe at position 15 preferred to be buried at the helical interface.

Figure 4

Mutations at position 15 of human IAPP affect the rate of fibril formation and the ability of insulin to inhibit this fibrillation. Residue substitutions computationally predicted to affect IAPP homodimerization and Insulin-IAPP heterodimerization were tested by a fibril formation assay. (A and B) Substitutions predicted to enhance helical association (F15A, F15S, F15D) of IAPP formed amyloid fibrils more rapidly than wild-type IAPP, as determined by Thioflavin T binding. The F15K mutation was predicted to disrupt the helical interface and a marked delay in fibril formation is observed. The 8–37 construct was used for both wild-type and Phe15 mutants. Mouse IAPP serves as a negative control for aggregation and never achieves a fluorescence value higher than baseline. (C) Including equimolar insulin in the assay delays the fibril formation of each mutant tested but has comparatively less of an inhibitory effect on the F15K substitution. (D) Representative EM images taken at 30 min of WT, F15S, and F15K to confirm the presence or absence of fibrils. Images of additional time points are included in the supporting Information. Scale bar represents 100 nm.

Figure 5

Model of nucleus formation of IAPP fibrils and the inhibition of fibrillation by insulin. (A) IAPP monomers are assumed to be in equilibrium between disordered state and helical, shown in (B) as it appears in the crystal. Phe15 is shown in stick view for reference. (C and D) Two IAPP dimers align their C-terminal amyloid domains in an antiparallel orientation. The C-terminus of each IAPP molecule is labeled. The position of residue S20 is indicated by the green arrow. When this residue is mutated to Glycine, fibrils form more rapidly. (E) This region, residues 23–37, forms the steric zipper spine of the fibril, by interdigitating sidechains with a second IAPP dimer. (F) Formation of the N-terminal strand occurs subsequent to spine formation. (G) Insulin prevents homodimeric helical association by binding to monomeric IAPP and blocking IAPP homodimerization. The insulin A-chain is shown in gold and the B chain is shown in gray.