Structural Characterisation of Fungal and Bacterial Amyloids by Hydrogen/Deuterium Exchange NMR Spectroscopy

In recent years, an increasing number of proteins has been shown to form amyloids that are involved in a large variety of cellular processes. Contrary to disease-related amyloids, these proteins fibrillise without toxic side effect into well ordered beta-sheet rich structures. In order to understand the formation and stability of these entities, well resolved structural data of amyloid fibrils are necessary. Recombinant fibrils of the HET-s prion domain from the filamentous fungus Fusarium graminearum FgHET-s(218-289) and the CsgA amyloid core of E. coli EcCsgA(40-151) were analysed by hydrogen/deuterium exchange NMR spectroscopy. The exchange rates demonstrated the formation of exceptionally stable and homogenous fibrils. In conjunction with solid state NMR experiments, the obtained exchange data revealed a close structural relationship of the HET-s homologues from Fusarium graminearum and Podospora anserina despite low sequence identity. Both prion domains constitute the same beta-solenoid fold enabling prion transmission between different species. The triangular hydrophobic core and major structural elements were identified to be conserved. Hydrogen/deuterium exchange analysis and thiol-labelling studies revealed a repetitive structural pattern of the amyloid core of EcCsgA with five imperfect repeats forming each a strand-turn-strand motif. Altogether EcCsgA(40-151) was suggested to fold into a beta-solenoid. Furthermore, heteronucleated EcCsgA fibrils were also subjected to hydrogen/deuterium exchange experiments. The corresponding experiments revealed that in vitro formed EcCsgB fibrils are no adequate seed. In order to analyse amyloid-like intermediates of EcCsgA, numerous fusion proteins were generated and analysed in consideration of their solubility and (non)amyloidogenic behaviour. At least two protein constructs were identified to be soluble and folded to a certain extend.