Virtual Quasi-2D Intermediates as Building Blocks for Plausible Structural Models of Amyloid Fibrils from Proteins with Complex Topologies: A Case Study of Insulin

[Image: see text] Conformational transitions of globular proteins into amyloid fibrils are complex multistage processes exceedingly challenging to simulate using molecular dynamics (MD). Slow monomer diffusion rates and rugged free energy landscapes disfavor swift self-assembly of orderly amyloid architectures within timescales accessible to all-atom MD. Here, we conduct a multiscale MD study of the amyloidogenic self-assembly of insulin: a small protein with a complex topology defined by two polypeptide chains interlinked by three disulfide bonds. To avoid kinetic traps, unconventional preplanarized insulin conformations are used as amyloid building blocks. These starting conformers generated through uniaxial compression of the native monomer in various spatial directions represent 6 distinct (out of 16 conceivable) two-dimensional (2D) topological classes varying in N-/C-terminal segments of insulin’s A- and B-chains being placed inside or outside of the central loop constituted by the middle sections of both chains and Cys7A–Cys7B/Cys19B–Cys20A disulfide bonds. Simulations of the fibrillar self-assembly are initiated through a biased in-register alignment of two, three, or four layers of flat conformers belonging to a single topological class. The various starting topologies are conserved throughout the self-assembly process resulting in polymorphic amyloid fibrils varying in structural features such as helical twist, presence of cavities, and overall stability. Some of the protofilament structures obtained in this work are highly compatible with the earlier biophysical studies on insulin amyloid and high-resolution studies on insulin-derived amyloidogenic peptide models postulating the presence of steric zippers. Our approach provides in silico means to study amyloidogenic tendencies and viable amyloid architectures of larger disulfide-constrained proteins with complex topologies.