Atomic resolution structure of full-length human insulin fibrils.

Patients with type 1 diabetes mellitus who are dependent on an external supply of insulin develop insulin-derived amyloidosis at the sites of insulin injection. A major component of these plaques is identified as full-length insulin consisting of the two chains A and B. While there have been several reports that characterize insulin misfolding and the biophysical properties of the fibrils, atomic-level information on the insulin fibril architecture remains elusive. We present here an atomic resolution structure of a monomorphic insulin amyloid fibril that has been determined using magic angle spinning solid-state NMR spectroscopy. The structure of the insulin monomer yields a U-shaped fold in which the two chains A and B are arranged in parallel to each other and are oriented perpendicular to the fibril axis. Each chain contains two ß-strands. We identify two hydrophobic clusters that together with the three preserved disulfide bridges define the amyloid core structure. The surface of the monomeric amyloid unit cell is hydrophobic implicating a potential dimerization and oligomerization interface for the assembly of several protofilaments in the mature fibril. The structure provides a starting point for the development of drugs that bind to the fibril surface and disrupt secondary nucleation as well as for other therapeutic approaches to attenuate insulin aggregation.

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.

Mechanistic insights into the aggregation pathway of the patient-derived immunoglobulin light chain variable domain protein FOR005.

Systemic antibody light chain (AL) amyloidosis is characterized by deposition of amyloid fibrils. Prior to fibril formation, soluble oligomeric AL protein has a direct cytotoxic effect on cardiomyocytes. We focus on the patient derived λ-III AL variable domain FOR005 which is mutated at five positions with respect to the closest germline protein. Using solution-state NMR spectroscopy, we follow the individual steps involved in protein misfolding from the native to the amyloid fibril state. Unfavorable mutations in the complementary determining regions introduce a strain in the native protein structure which yields partial unfolding. Driven by electrostatic interactions, the protein converts into a high molecular weight, oligomeric, molten globule. The high local concentration of aggregation prone regions in the oligomer finally catalyzes the conversion into fibrils. The topology is determined by balanced electrostatic interactions in the fibril core implying a 180° rotational switch of the beta-sheets around the conserved disulfide bond.

Field and magic angle spinning frequency dependence of proton resonances in rotating solids.

Proton detection in solid state NMR is continuously developing and allows one to gain new insights in structural biology. Overall, this progress is a result of the synergy between hardware development, new NMR methodology and new isotope labeling strategies, to name a few factors. Even though current developments are rapid, it is worthwhile to summarize what can currently be achieved employing proton detection in biological solids. We illustrate this by analysing the signal-to-noise ratio (SNR) for spectra obtained for a microcrystalline α-spectrin SH3 domain protein sample by (i) employing different degrees of chemical dilution to replace protons by incorporating deuterons in different sites, by (ii) variation of the magic angle spinning (MAS) frequencies between 20 and 110 kHz, and by (iii) variation of the static magnetic field B0. The experimental SNR values are validated with numerical simulations employing up to 9 proton spins. Although in reality a protein would contain far more than 9 protons, in a deuterated environment this is a sufficient number to achieve satisfactory simulations consistent with the experimental data. The key results of this analysis are (i) with current hardware, deuteration is still necessary to record spectra of optimum quality; (ii) 13CH3 isotopomers for methyl groups yield the best SNR when MAS frequencies above 100 kHz are available; and (iii) sensitivity increases with a factor beyond B0 3/2 with the static magnetic field due to a transition of proton-proton dipolar interactions from a strong to a weak coupling limit.

SAA fibrils involved in AA amyloidosis are similar in bulk and by single particle reconstitution: A MAS solid-state NMR study.

AA amyloidosis is one of the most prevalent forms of systemic amyloidosis and affects both humans and other vertebrates. In this study, we compare MAS solid-state NMR data with a recent cryo-EM study of fibrils involving full-length murine SAA1.1. We address the question whether the specific requirements for the reconstitution of an amyloid fibril structure by cryo-EM can potentially yield a bias towards a particular fibril polymorph. We employ fibril seeds extracted from in to vivo material to imprint the fibril structure onto the biochemically produced protein. Sequential assignments yield the secondary structure elements in the fibril state. Long-range DARR and PAR experiments confirm largely the topology observed in the ex-vivo cryo-EM study. We find that the ß-sheets identified in the NMR experiments are similar to the ß-sheets found in the cryo-EM study, with the exception of amino acids 33-42. These residues cannot be assigned by solid-state NMR, while they adopt a stable ß-sheet in the cryo-EM structure. We suggest that the differences between MAS solid-state NMR and cryo-EM data are a consequence of a second conformer involving residues 33-42. Moreover, we were able to characterize the dynamic C-terminal tail of SAA in the fibril state. The C-terminus is flexible, remains detached from the fibrils, and does not affect the SAA fibril structure as confirmed further by molecular dynamics simulations. As the C-terminus can potentially interact with other cellular components, binding to cellular targets can affect its accessibility for protease digestion.

Structural insights into the interaction of antifungal peptides and ergosterol containing fungal membrane.

The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.

Solid state NMR assignments of a human λ-III immunoglobulin light chain amyloid fibril.

The aggregation of antibody light chains is linked to systemic light chain (AL) amyloidosis, a disease where amyloid deposits frequently affect the heart and the kidney. We here investigate fibrils from the λ-III FOR005 light chain (LC), which is derived from an AL-patient with severe cardiac involvement. In FOR005, five residues are mutated with respect to its closest germline gene segment IGLV3-19 and IGLJ3. All mutations are located close to the complementarity determining regions (CDRs). The sequence segments responsible for the fibril formation are not yet known. We use fibrils extracted from the heart of this particular amyloidosis patient as seeds to prepare fibrils for solid-state NMR. We show that the seeds induce the formation of a specific fibril structure from the biochemically produced protein. We have assigned the fibril core region of the FOR005-derived fibrils and characterized the secondary structure propensity of the observed amino acids. As the primary structure of the aggregated patient protein is different for every AL patient, it is important to study, analyze and report a greater number of light chain sequences associated with AL amyloidosis.

Seeded fibrils of the germline variant of human λ-III immunoglobulin light chain FOR005 have a similar core as patient fibrils with reduced stability.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt ß-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

Structural Insight into IAPP-Derived Amyloid Inhibitors and Their Mechanism of Action.

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross-amyloid interaction surface with Aß (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aß amyloid self-assembly. However, the molecular mechanism of their function is not well understood. Using solution-state and solid-state NMR spectroscopy in combination with ensemble-averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3-GI is highly dynamic, can adopt a ß-like structure, and oligomerizes into colloid-like assemblies in a process that is reminiscent of liquid-liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aß40. Sequestration of substrates into these colloid-like structures provides a mechanistic basis for ISM function and the design of novel potent anti-amyloid molecules.

Stabilization and structural analysis of a membrane-associated hIAPP aggregation intermediate.

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique ß-strand structure distinct from the conventional amyloid ß-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

The redox environment triggers conformational changes and aggregation of hIAPP in Type II Diabetes.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in ß-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

Sulindac Sulfide Induces the Formation of Large Oligomeric Aggregates of the Alzheimer's Disease Amyloid-ß Peptide Which Exhibit Reduced Neurotoxicity.

Alzheimer's disease is characterized by deposition of the amyloid ß-peptide (Aß) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the nonsteroidal anti-inflammatory drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aß peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation. In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aß solution. We find that sulindac sulfide induced Aß aggregates are structurally homogeneous. The C-terminal part of the peptide adopts a ß-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in wild type fibril structures. (13)C-(19)F transferred echo double resonance experiments suggest that sulindac sulfide colocalizes with the Aß peptide in the aggregate.