Structural complexity of a composite amyloid fibril

Amyloid fibrils represent an aggregated state of proteins that appears structurally conserved even in presence of substantial differences in protein primary sequence. In this context a very common motif is the elegant and seemingly universal serpentine parallel in-register alignment of repeating identically structured monomers. An early high-resolution view of such a structure was obtained on amyloid-like crystals of a peptide fragment from yeast prion protein Sup35p (GNNQQNY7-13). As GNNQQNY also forms amyloid-like fibrils under similar conditions, it has been theorized that the crystal's structural features are shared by amyloid-like fibrils. Here we apply magic-angle-spinning (MAS) solid-state NMR to probe the structure and dynamics of the actual GNNQQNY fibrils. Multiple NMR signals were previously observed for such samples, seemingly consistent with the presence of polymorphic fibrils. Here we demonstrate that peptides with these three distinct conformations instead assemble together into a composite protofilament structure. Intriguingly, electron-microscopy (EM) shows ribbon-like fibrillar assemblies with variable striation widths, suggesting that protofilaments are present in different types of supramolecular configurations. Structural and dynamical NMR data reveal the presence of highly restricted side chain conformations involved in interfaces between differently structured peptides, likely comprising interdigitated steric zippers. We outline molecular interfaces that are consistent with the observed EM and NMR data. The rigid and uniform structure of the GNNQQNY crystals is found to contrast distinctly with the more complex structural and dynamic nature of these "composite" amyloid fibrils. These results provide insight into the fibril-crystal distinction and also indicate a necessary caution with respect to the extrapolation of crystal structures to the study of fibril structure and formation.