A Helical Structural Nucleus Is the Primary Elongating Unit of Insulin Amyloid Fibrils

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Abstract
Although amyloid fibrillation is generally believed to be a nucleation-dependent process, the nuclei are largely structurally uncharacterized. This is in part due to the inherent experimental challenge associated with structural descriptions of individual components in a dynamic multi-component equilibrium. There are indications that oligomeric aggregated precursors of fibrillation, and not mature fibrils, are the main cause of cytotoxicity in amyloid disease. This further emphasizes the importance of characterizing early fibrillation events. Here we present a kinetic x-ray solution scattering study of insulin fibrillation, revealing three major components: insulin monomers, mature fibrils, and an oligomeric species. Low-resolution three-dimensional structures are determined for the fibril repeating unit and for the oligomer, the latter being a helical unit composed of five to six insulin monomers. This helical oligomer is likely to be a structural nucleus, which accumulates above the supercritical concentration used in our experiments. The growth rate of the fibrils is proportional to the amount of the helical oligomer present in solution, suggesting that these oligomers elongate the fibrils. Hence, the structural nucleus and elongating unit in insulin amyloid fibrillation may be the same structural component above supercritical concentrations. A novel elongation pathway of insulin amyloid fibrils is proposed, based on the shape and size of the fibrillation precursor. The distinct helical oligomer described in this study defines a conceptually new basis of structure-based drug design against amyloid diseases. Diseases associated with the presence of amyloid structures, such as Alzheimer and Parkinson disease, are characterized by the presence of protein aggregates in the form of highly ordered fibrils. This amyloid fibril formation is also commonly observed for a number of protein drugs, such as insulin. Detailed information on how and why these fibrils are formed will be very useful to design compounds and drugs that may reverse or even prevent fibril formation, but existing knowledge in this field is still limited. We have studied, in real time, the fibril formation of insulin using a technique based on scattering of x-rays (small-angle x-ray scattering [SAXS]). Using SAXS, we obtained hitherto unprecedented three-dimensional structural information on these fibrils in solution. Most importantly, we were able to describe the three-dimensional structure of a crucial intermediate, which is probably a structural starting point (nucleus) in the fibril formation process. These results suggest that under our experimental conditions this crucial intermediate serves both as the fibrillation nucleus, as well as the elongating species. We propose that the latter intermediate is an interesting target for small molecules in order to prevent or reduce amyloid fibril formation.