NMR Solution Structure of an Invisible Protein State at the Edge between Folding and Aggregation into Amyloid Fibrils

摘要

Protein folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. To gain insight, we used CPMG Relaxation dispersion NMR spectroscopy to determine the atomic-resolution three-dimensional solution structure of a 2% populated, on-pathway folding intermediate of the A39V/N53P/V55L Fyn SH3 domain. To this end, we used the backbone chemical shifts and RDCs/RCSAs of the “invisible” intermediate reconstructed from CPMG experiments as experimental input for structure calculations based on chemical shift restrained replica exchange molecular dynamics simulations via the CamShift approach (1). The COOH-terminus remains disordered in this intermediate (2), thereby exposing the aggregation-prone NH 2-terminal beta-strand. Accordingly, mutants lacking the COOH-terminus and thus mimicking the intermediatea fail to safeguard the folding route and spontaneously form β-sheet-rich fibrillar aggregates with a diameter of several nanometers and an affinity for the dye Congo red. The structure provides a detailed characterization of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.