Mechanism of islet amyloid polypeptide fibrillation at lipid interfaces studied by infrared reflection absorption spectroscopy

Abstract

Islet amyloid polypeptide (IAPP) is a pancreatic hormone and one of a number of proteins that are involved in the formation of amyloid deposits in the islets of Langerhans of type II diabetes mellitus patients. Though IAPP-membrane interactions are known to play a major role in the fibrillation process, the mechanism and the peptide's conformational changes involved are still largely unknown. To obtain new insights into the conformational dynamics of IAPP upon its aggregation at membrane interfaces and to relate these structures to its fibril formation, we studied the association of IAPP at various interfaces including neutral as well as charged phospholipids using infrared reflection absorption spectroscopy. The results obtained reveal that the interaction of human IAPP with the lipid interface is driven by the N-terminal part of the peptide and is largely driven by electrostatic interactions, as the protein is able to associate strongly with negatively charged lipids only. A two-step process is observed upon peptide binding, involving a conformational transition from a largely alpha-helical to a beta-sheet conformation, finally forming ordered fibrillar structures. As revealed by simulations of the infrared reflection absorption spectra and complementary atomic force microscopy studies, the fibrillar structures formed consist of parallel intermolecular beta-sheets lying parallel to the lipid interface but still contain a significant number of turn structures. We may assume that these dynamical conformational changes observed for negatively charged lipid interfaces play an important role as the first steps of IAPP-induced membrane damage in type II diabetes.

SCHEME 1

Primary sequences of human and rIAPP. The amino acid residues marked in light gray belong to the “amyloidogenic core” proposed for IAPP. The six amino acid residues drawn in dark gray highlight the differences between the two sequences.

FIGURE 1

(A) Surface-pressure versus time course of hIAPP film adsorbed at the water-air interface (1 μM hIAPP subphase concentration) and (B) IRRA spectra of the hIAPP film at the respective positions of the surface-pressure versus time curve given in A.

FIGURE 2

(A) Surface-pressure versus time course of a POPG film with 1 μM hIAPP and (B) IRRA spectra of the amide I and II regions for POPG monolayer starting at 10 mN/m at the respective positions of the surface-pressure versus time curve given in A. (C) Surface-pressure versus time course of a POPG film with 1 μM hIAPP and (D) IRRA spectra of the amide I and II regions for POPG monolayer starting at 30 mN/m at the respective positions of the surface-pressure versus time curve given in C. All IRRA spectra were acquired using p-polarized light at an angle of incidence of 40°.

FIGURE 3

(A) Time dependence of the intensity of the amide I band region of 1 μM hIAPP at the POPG monolayer at a surface pressure of 10 mN/m. (B) Corresponding changes of the secondary structure elements. Owing to unknown transition dipole moments, only relative changes should be discussed.

FIGURE 4

(A) Surface-pressure versus time course of a POPG film with 1 μM rIAPP and (B) IRRA spectra of the lipid film at the respective positions of the surface-pressure versus time curve given in A (please note the drastically expanded intensity scale). All spectra were recorded at an angle of incidence of 40° with p-polarized light. (C) IRRA spectra of 1 μM hIAPP adsorbed at the water-POPG monolayer interface acquired with p-polarized light at various incident angles and a surface pressure of 32.2 mN/m. (D) Simulations of IRRA spectra of a β-sheet lying flat at the water-air interface. The calculation was performed for p-polarized light and different angles of incidence for the amide I and II band regions.

SCHEME 2

Representation of secondary structure predictions for hIAPP (algorithms used (,–58): 1: double prediction; 2: hierarchical neural network classifiers; 3: self-optimized prediction method).

FIGURE 5

(A) AFM image of the aggregate structure of IAPP (1 μM), formed at and taken from underneath the POPG interface and deposited on freshly cleaved mica. Filaments with a width of ∼70 nm and a height of ∼4 nm are found. (B) AFM image of a sample of rIAPP (1 μM), taken from the POPG interface after 20 h. Some small oligomeric or amorphous structures with a height of ∼3 nm are seen only.

FIGURE 6

Schematic model of hIAPP-POPG monolayer interaction, lipid-induced hIAPP conformational transitions, and fibril formation. According to Kajava et al., upon fibril formation, serpentine-like superpleated β-structural elements of IAPP may be assumed to form that stack in register, with a 0.47 nm axial rise and a small rotational twist per step, thus generating an array of parallel β-sheets (66).