Catalysis driven by an amyloid-substrate complex.

Amine modification through nucleophilic attack of the amine functionality is a very common chemical transformation. Under biorelevant conditions using acidic-to-neutral pH buffer, however, the nucleophilic reaction of alkyl amines (pKa ≈ 10) is not facile due to the generation of ammonium ions lacking nucleophilicity. Here, we disclose a unique molecular transformation system, catalysis driven by amyloid-substrate complex (CASL), that promotes amine modifications in acidic buffer. Ammonium ions attached to molecules with amyloid-binding capability were activated through deprotonation due to the close proximity to the amyloid catalyst formed by Ac-Asn-Phe-Gly-Ala-Ile-Leu-NH2 (NL6), derived from islet amyloid polypeptide (IAPP). Under the CASL conditions, alkyl amines underwent various modifications, i.e., acylation, arylation, cyclization, and alkylation, in acidic buffer. Crystallographic analysis and chemical modification studies of the amyloid catalysts suggested that the carbonyl oxygen of the Phe-Gly amide bond of NL6 plays a key role in activating the substrate amine by forming a hydrogen bond. Using CASL, selective conversion of substrates possessing equivalently reactive amine functionalities was achieved in catalytic reactions using amyloids. CASL provides a unique method for applying nucleophilic conversion reactions of amines in diverse fields of chemistry and biology.

Structural Insights into Seeding Mechanisms of hIAPP Fibril Formation.

The deposition of islet amyloid polypeptide (hIAPP) fibrils is a hallmark of ß-cell death in type II diabetes. In this study, we employ state-of-the-art MAS solid-state spectroscopy to investigate the previously elusive N-terminal region of hIAPP fibrils, uncovering both rigidity and heterogeneity. Comparative analysis between wild-type hIAPP and a disulfide-deficient variant (hIAPPC2S,C7S) unveils shared fibril core structures yet strikingly distinct dynamics in the N-terminus. Specifically, the variant fibrils exhibit extended ß-strand conformations, facilitating surface nucleation. Moreover, our findings illuminate the pivotal roles of specific residues in modulating secondary nucleation rates. These results deepen our understanding of hIAPP fibril assembly and provide critical insights into the molecular mechanisms underpinning type II diabetes, holding promise for future therapeutic strategies.

Neuroinflammation induced by amyloid-forming pancreatic amylin: Rationale for a mechanistic hypothesis.

Amylin is a systemic neuroendocrine hormone co-expressed and co-secreted with insulin by pancreatic ß-cells. In persons with thype-2 diabetes, amylin forms pancreatic amyloid triggering inflammasome and interleukin-1ß signaling and inducing ß-cell apoptosis. Here, we summarize recent progress in understanding the potential link between amyloid-forming pancreatic amylin and Alzheimer's disease (AD). Clinical data describing amylin pathology in AD alongside mechanistic studies in animals are reviewed. Data from multiple research teams indicate higher amylin concentrations are associated with increased frequency of cognitive impairment and amylin co-aggregates with ß-amyloid in AD-type dementia. Evidence from rodent models further suggests cerebrovascular amylin accumulation as a causative factor underlying neurological deficits. Analysis of relevant literature suggests that modulating the amylin-interleukin-1ß pathway may provide an approach for counteracting neuroinflammation in AD.

Experimental and computational investigation of the effect of Hsc70 structural variants on inhibiting amylin aggregation.

The misfolding and aggregation of human islet amyloid polypeptide (hIAPP), also known as amylin, have been implicated in the pathogenesis of type 2 diabetes (T2D). Heat shock proteins, specifically, heat shock cognate 70 (Hsc70), are molecular chaperones that protect against hIAPP misfolding and inhibits its aggregation. Nevertheless, there is an incomplete understanding of the mechanistic interactions between Hsc70 domains and hIAPP, thus limiting their potential therapeutic role in diabetes. This study investigates the inhibitory capacities of different Hsc70 variants, aiming to identify the structural determinants that strike a balance between efficacy and cytotoxicity. Our experimental findings demonstrate that the ATPase activity of Hsc70 is not a pivotal factor for inhibiting hIAPP misfolding. We underscore the significance of the C-terminal substrate-binding domain of Hsc70 in inhibiting hIAPP aggregation, emphasizing that the removal of the lid subdomain diminishes the inhibitory effect of Hsc70. Additionally, we employed atomistic discrete molecular dynamics simulations to gain deeper insights into the interaction between Hsc70 variants and hIAPP. Integrating both experimental and computational findings, we propose a mechanism by which Hsc70's interaction with hIAPP monomers disrupts protein-protein connections, primarily by shielding the ß-sheet edges of the Hsc70-ß-sandwich. The distinctive conformational dynamics of the alpha helices of Hsc70 potentially enhance hIAPP binding by obstructing the exposed edges of the ß-sandwich, particularly at the ß5-ß8 region along the alpha helix interface. This, in turn, inhibits fibril growth, and similar results were observed following hIAPP dimerization. Overall, this study elucidates the structural intricacies of Hsc70 crucial for impeding hIAPP aggregation, improving our understanding of the potential anti-aggregative properties of molecular chaperones in diabetes treatment.

Development of a Novel Assay for Direct Assessment of Selective Amylin Receptor Activation Reveals Novel Differences in Behavior of Selective and Nonselective Peptide Agonists.

Dual amylin and calcitonin receptor agonists (DACRAs) show promise as efficacious therapeutics for treatment of metabolic disease, including obesity. However, differences in efficacy in vivo have been observed for individual DACRAs, indicating that detailed understanding of the pharmacology of these agents across target receptors is required for rational drug development. To date, such understanding has been hampered by lack of direct, subtype-selective, functional assays for the amylin receptors (AMYRs). Here, we describe the generation of receptor-specific assays for recruitment of Venus-tagged Gs protein through fusion of luciferase to either the human calcitonin receptor (CTR), human receptor activity-modifying protein (RAMP)-1, RAMP1 (AMY1R), human RAMP2 (AMY2R), or human RAMP3 (AMY3R). These assays revealed a complex pattern of receptor activation by calcitonin, amylin, or DACRA peptides that was distinct at each receptor subtype. Of particular note, although both of the CT-based DACRAs, sCT and AM1784, displayed relatively similar behaviors at CTR and AMY1R, they generated distinct responses at AMY2R and AMY3R. These data aid the rationalization of in vivo differences in response to DACRA peptides in rodent models of obesity. Direct assessment of the pharmacology of novel DACRAs at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases. SIGNIFICANCE STATEMENT: Amylin receptors (AMYRs) are important obesity targets. Here we describe a novel assay that allows selective functional assessment of individual amylin receptor subtypes that provides unique insight into the pharmacology of potential therapeutic ligands. Direct assessment of the pharmacology of novel agonists at AMYR subtypes is likely to be important for development of optimized therapeutics for treatment of metabolic diseases.

Amylin in Alcohol Addiction: A Potential New Treatment Target or an Adjuvant to Other Treatments?

Amylin is a neuroendocrine hormone with a potential role in addictive disorders, including alcohol use disorder (AUD). In addition to reducing appetitive behavior, amylin has been shown to affect alcohol-related behaviors in rodents. Delineating the biobehavioral correlates of amylin in relation to alcohol seeking and consumption has the potential of identifying new treatment targets for AUD, yet additional translational and human research is needed.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded ß-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Chronic pramlintide decreases feeding via a reduction in meal size in male rats.

Amylin, a pancreatic hormone, is well-established to suppress feeding by enhancing satiation. Pramlintide, an amylin analog that is FDA-approved for the treatment of diabetes, has also been shown to produce hypophagia. However, the behavioral mechanisms underlying the ability of pramlintide to suppress feeding are unresolved. We hypothesized that systemic pramlintide administration in rats would reduce energy intake, specifically by reducing meal size. Male rats were given b.i.d. administration of intraperitoneal pramlintide or vehicle for 1 week, and chow intake, meal patterns, and body weight were monitored throughout the test period. Consistent with our hypothesis, pramlintide decreased chow intake mainly via suppression of meal size, with corresponding reductions in meal duration on several days. Fewer effects on meal number or feeding rate were detected. Pramlintide also reduced weight gain over the 1-week study. These results highlight that the behavioral mechanisms by which pramlintide produces hypophagia are similar to those driven by amylin itself, and provide important insight into the ability of this pharmacotherapy to promote negative energy balance over a period of chronic administration.

Liraglutide versus pramlintide in protecting against cognitive function impairment through affecting PI3K/AKT/GSK-3ß/TTBK1 pathway and decreasing Tau hyperphosphorylation in high-fat diet- streptozocin rat model.

The American Diabetes Association guidelines (2021) confirmed the importance of raising public awareness of diabetes-induced cognitive impairment, highlighting the links between poor glycemic control and cognitive impairment. The characteristic brain lesions of cognitive dysfunction are neurofibrillary tangles (NFT) and senile plaques formed of amyloid-ß deposition, glycogen synthase kinase 3 beta (GSK3ß), and highly homologous kinase tau tubulin kinase 1 (TTBK1) can phosphorylate Tau proteins at different sites, overexpression of these enzymes produces extensive phosphorylation of Tau proteins making them insoluble and enhance NFT formation, which impairs cognitive functions. The current study aimed to investigate the potential contribution of liraglutide and pramlintide in the prevention of diabetes-induced cognitive dysfunction and their effect on the PI3K/AKT/GSK-3ß/TTBK1 pathway in type 2 diabetic (T2D) rat model. T2D was induced by administration of a high-fat diet for 10 weeks, then injection of a single dose of streptozotocin (STZ); treatment was started with either pramlintide (200 µg/kg/day sc) or liraglutide (0.6 mg/kg/day sc) for 6 weeks in addition to the HFD. At the end of the study, cognitive functions were assessed by novel object recognition and T-maze tests. Then, rats were sacrificed for biochemical and histological assessment of the hippocampal tissue. Both pramlintide and liraglutide treatment revealed equally adequate control of diabetes, prevented the decline in memory function, and increased PI3K/AKT expression while decreasing GSK-3ß/TTBK1 expression; however, liraglutide significantly decreased the number of Tau positive cells better than pramlintide did. This study confirmed that pramlintide and liraglutide are promising antidiabetic medications that could prevent associated cognitive disorders in different mechanisms.

Calcitonin receptor, calcitonin gene-related peptide and amylin distribution in C1/2 dorsal root ganglia.

BACKGROUND: The upper cervical dorsal root ganglia (DRG) are important for the transmission of sensory information associated with the back of the head and neck, contributing to head pain. Calcitonin receptor (CTR)-based receptors, such as the amylin 1 (AMY1) receptor, and ligands, calcitonin gene-related peptide (CGRP) and amylin, have been linked to migraine and pain. However, the contribution of this system to nociception involving the cervical DRG is unclear. Therefore, this study aimed to determine the relative distribution of the CTR, CGRP, and amylin in upper cervical DRG. METHODS: CTR, CGRP, and amylin immunofluorescence was examined relative to neural markers in C1/2 DRG from male and female mice, rats, and human cases. Immunofluorescence was supported by RNA-fluorescence in situ hybridization examining amylin mRNA distribution in rat DRG. RESULTS: Amylin immunofluorescence was observed in neuronal soma and fibres. Amylin mRNA (Iapp) was also detected. Amylin and CGRP co-expression was observed in 19% (mouse), 17% (rat), and 36% (human) of DRG neurons in distinct vesicle-like neuronal puncta from one another. CTR immunoreactivity was present in DRG neurons, and both peptides produced receptor signalling in primary DRG cell cultures. CTR-positive neurons frequently co-expressed amylin and/or CGRP (66% rat; 84% human), with some sex differences. CONCLUSIONS: Amylin and CGRP could both be local peptide agonists for CTR-based receptors in upper cervical DRG, potentially acting through autocrine and/or paracrine signalling mechanisms to modulate neuron function. Amylin and its receptors could represent novel pain targets.

The processing intermediate of human amylin, pro-amylin(1-48), has in vivo and in vitro bioactivity.

Amylin is released by pancreatic beta-cells in response to a meal and its major soluble mature form (37 amino acid-peptide) produces its biological effects by activating amylin receptors. Amylin is derived from larger propeptides that are processed within the synthesizing beta-cell. There are suggestions that a partially processed form, pro-amylin(1-48) is also secreted. We tested the hypothesis that pro-amylin(1-48) has biological activity and that human pro-amylin(1-48) may also form toxic pre-amyloid species. Amyloid formation, the ability to cross-seed and in vitro toxicity were similar between human pro-amylin(1-48) and amylin. Human pro-amylin(1-48) was active at amylin-responsive receptors, though its potency was reduced at rat, but not human amylin receptors. Pro-amylin(1-48) was able to promote anorexia by activating neurons of the area postrema, amylin's primary site of action, indicating that amylin can tolerate significant additions at the N-terminus without losing bioactivity. Our studies help to shed light on the possible roles of pro-amylin(1-48) which may be relevant for the development of future amylin-based drugs.

Are insulin sensitizers the new strategy to treat Type 1 diabetes? A long-acting dual amylin and calcitonin receptor agonist improves insulin-mediated glycaemic control and controls body weight.

BACKGROUND AND PURPOSE: Insulin therapies for Type 1 diabetes (T1D) have limitations, such as glucose fluctuations, hypoglycaemia, and weight gain. Only pramlintide is approved with insulin. However, its short half-life limits efficacy, requiring multiple daily injections and increasing hypoglycaemia risk. New strategies are needed to improve glycaemic control. Dual amylin and calcitonin receptor agonists are potent insulin sensitizers developed for Type 2 diabetes (T2D) as they improve glucose control, reduce body weight, and attenuate hyperglucagonemia. However, it is uncertain if they could be used to treat T1D. EXPERIMENTAL APPROACH: Sprague Dawley rats received a single intravenous injection of streptozotocin (STZ) (50 mg·kg-1) to induce T1D. Humulin (1 U/200 g·day-1 or 2 U/200 g·day-1) was continuously infused, while half of the rats received additional KBP-336 (4.5 nmol·kg-1 Q3D) treatment. Bodyweight, food intake, and blood glucose were monitored throughout the study. An oral glucose tolerance test was performed during the study. KEY RESULTS: Treatment with Humulin or Humulin + KBP-336 improved the health of STZ rats. Humulin increased body weight in STZ rats, but KBP-336 attenuated these increases and maintained a significant weight loss. The combination exhibited greater blood glucose reductions than Humulin-treated rats alone, reflected by improved HbA1c levels and glucose control. The combination prevented hyperglucagonemia, reduced amylin levels, and increased pancreatic insulin content, indicating improved insulin sensitivity and beta-cell preservation. CONCLUSION AND IMPLICATIONS: The insulin sensitizer KBP-336 lowered glucagon secretion while attenuating insulin-induced weight gain. Additionally, KBP-336 may prevent hypoglycaemia and improve insulin resistance, which could be a significant advantage for individuals with T1D seeking therapeutic benefits.

Analyzing Gene Expression After Administration of Low-Intensity Therapeutic Ultrasound in Human Islet Cells.

OBJECTIVES: Diabetes mellitus is a complex heterogenous metabolic disease that significantly affects the world population. Although many treatments exist, including medications such as metformin, sulfonylureas, and glucagon-like peptide-1 (GLP) receptor agonist, there is growing interest in finding alternative methods to noninvasively treat this disease. It has been previously shown that low-intensity ultrasound stimulation of pancreatic ß-cells in mice can elicit insulin secretion as a potential treatment for this disease. This is desirable as therapeutic ultrasound has the ability to induce bioeffects while selectively focusing deep within tissues, allowing for modulation of hormone secretion in the pancreas to mitigate insufficient levels of insulin. METHODS: Exactly 800 kHz ultrasound with intensity 0.5 W/cm2 was administered 5 minutes continuously, that is, 100% duty cycle, to donor pancreatic human islets, followed by 1 hour incubation and RT-qPCR to assess the effect of ultrasound stimulation on gene expression. The genes were insulin (INS), glucagon (Glu), amylin (Amy), and binding immunoglobulin protein (BiP). Nine donor pancreatic human islets were used to assess insulin and glucagon secretion, while eight samples were used for amylin and BiP. Fold change (FC) was calculated to analyze the effect of ultrasound stimulation on the gene expression of the donor islet cells. High-glucose and thapsigargin-treated islets were utilized as positive controls. Cell viability testing was done using a Trypan Blue Exclusion Test. RESULTS: Ultrasound stimulation did not cause a statistically significant upregulation in any of the tested genes (INS FC = 1.15, P-value = .5692; Glu FC = 1.60, P-value = .2231; Amy FC, P-value = .2863; BiP FC = 2.68, P-value = .3907). CONCLUSIONS: The results of this study show that the proposed ultrasound treatment parameters do not appear to significantly affect gene expression of any gene tested.

Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins.

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Robust Chemical Synthesis of "Difficult Peptides" via 2-Hydroxyphenol-pseudoproline (ψ2-hydroxyphenolpro) Modifications.

The challenging preparation of "difficult peptides" has always hindered the development of peptide-active pharmaceutical ingredients. Pseudoproline (ψpro) building blocks have been proven effective and powerful tools for the synthesis of "difficult peptides". In this paper, we efficiently prepared a set of novel 2-(oxazolidin-2-yl)phenol compounds as proline surrogates (2-hydroxyphenol-pseudoprolines, ψ2-hydroxyphenolpro) and applied it in the synthesis of many well-known "difficult peptides", including human thymosin α1, amylin, and ß-amyloid (1-42) (Aß42).

Multiscale Modeling of Macromolecular Interactions between Tau-Amylin Oligomers and Asymmetric Lipid Nanodomains That Link Alzheimer's and Diabetic Diseases.

The molecular events of protein misfolding and self-aggregation of tau and amylin are associated with the progression of Alzheimer's and diabetes, respectively. Recent studies suggest that tau and amylin can form hetero-tau-amylin oligomers. Those hetero-oligomers are more neurotoxic than homo-tau oligomers. So far, the detailed interactions between the hetero-oligomers and the neuronal membrane are unknown. Using multiscale MD simulations, the lipid binding and protein folding behaviors of hetero-oligomers on asymmetric lipid nanodomains or raft membranes were examined. Our raft membranes contain phase-separated phosphatidylcholine (PC), cholesterol, and anionic phosphatidylserine (PS) or ganglioside (GM1) in one leaflet of the lipid bilayer. The hetero-oligomers bound more strongly to the PS and GM1 than other lipids via the hydrophobic and hydrophilic interactions, respectively, in the raft membranes. The hetero-tetramer disrupted the acyl chain orders of both PC and PS in the PS-containing raft membrane, but only the GM1 in the GM1-containing raft membrane as effectively as the homo-tau-tetramer. We discovered that the alpha-helical content in the heterodimer was greater than the sum of alpha-helical contents from isolated tau and amylin monomers on both raft membranes, indicative of a synergetic effect of tau-amylin interactions in surface-induced protein folding. Our results provide new molecular insights into understanding the cross-talk between Alzheimer's and diabetes.

The Inhibition Effect of Epigallocatechin-3-Gallate on the Co-Aggregation of Amyloid-ß and Human Islet Amyloid Polypeptide Revealed by Replica Exchange Molecular Dynamics Simulations.

Alzheimer's disease and Type 2 diabetes are two epidemiologically linked diseases which are closely associated with the misfolding and aggregation of amyloid proteins amyloid-ß (Aß) and human islet amyloid polypeptide (hIAPP), respectively. The co-aggregation of the two amyloid proteins is regarded as the fundamental molecular mechanism underlying their pathological association. The green tea extract epigallocatechin-3-gallate (EGCG) has been extensively demonstrated to inhibit the amyloid aggregation of Aß and hIAPP proteins. However, its potential role in amyloid co-aggregation has not been thoroughly investigated. In this study, we employed the enhanced-sampling replica exchange molecular dynamics simulation (REMD) method to investigate the effect of EGCG on the co-aggregation of Aß and hIAPP. We found that EGCG molecules substantially diminish the ß-sheet structures within the amyloid core regions of Aß and hIAPP in their co-aggregates. Through hydrogen-bond, π-π and cation-π interactions targeting polar and aromatic residues of Aß and hIAPP, EGCG effectively attenuates both inter-chain and intra-chain interactions within the co-aggregates. All these findings indicated that EGCG can effectively inhibit the co-aggregation of Aß and hIAPP. Our study expands the potential applications of EGCG as an anti-amyloidosis agent and provides therapeutic options for the pathological association of amyloid misfolding disorders.

Amylin, Another Important Neuroendocrine Hormone for the Treatment of Diabesity.

Diabetes mellitus is a devastating chronic metabolic disease. Since the majority of type 2 diabetes mellitus patients are overweight or obese, a novel term-diabesity-has emerged. The gut-brain axis plays a critical function in maintaining glucose and energy homeostasis and involves a variety of peptides. Amylin is a neuroendocrine anorexigenic polypeptide hormone, which is co-secreted with insulin from ß-cells of the pancreas in response to food consumption. Aside from its effect on glucose homeostasis, amylin inhibits homeostatic and hedonic feeding, induces satiety, and decreases body weight. In this narrative review, we summarized the current evidence and ongoing studies on the mechanism of action, clinical pharmacology, and applications of amylin and its analogs, pramlintide and cagrilintide, in the field of diabetology, endocrinology, and metabolism disorders, such as obesity.

Structural diversity in the membrane-bound hIAPP dimer correlated with distinct membrane disruption mechanisms.

Amyloid deposits of the human islet amyloid polypeptide (hIAPP) have been identified in 90% of patients with type II diabetes. Cellular membranes accelerate the hIAPP fibrillation, and the integrity of membranes is also disrupted at the same time, leading to the apoptosis of ß cells in pancreas. The molecular mechanism of hIAPP-induced membrane disruption, especially during the initial membrane disruption stage, has not been well understood yet. Herein, we carried out extensive all-atom molecular dynamics simulations investigating the hIAPP dimerization process in the anionic POPG membrane, to provide the detailed molecular mechanisms during the initial hIAPP aggregation stage in the membrane environment. Compared to the hIAPP monomer on the membrane, we observed not only an increase of α-helical structures, but also a substantial increase of ß-sheet structures upon spontaneous dimerization. Moreover, the random coiled and α-helical dimer structures insert deep into the membrane interior with a few inter-chain contacts at the C-terminal region, while the ß-sheet-rich structures reside on the membrane surface accompanied by strong inter-chain hydrophobic interactions. The coexistence of α and ß structures constitutes a diverse structural ensemble of the membrane-bound hIAPP dimer. From α-helical to ß-sheet structures, the degree of membrane disruption decreases gradually, and thus the membrane damage induced by random coiled and α-helical structures precedes that induced by ß-sheet structures. We speculate that insertion of random coiled and α-helical structures contributes to the initial stage of membrane damage, while ß-sheet structures on the membrane surface are more involved in the later stage of fibril-induced membrane disruption.