Amyloid Forming Human Lysozyme Intermediates are Stabilized by Non-Native Amide-π Interactions.

Mutational variants of human lysozyme cause a rare but fatal hereditary systemic amyloidosis by populating an intermediate state that self-assembles into amyloid fibrils. However, this intermediate state is recalcitrant to detailed structural investigation, as it is only transiently and sparsely populated. Here, this work investigates the intermediate state of an amyloid-forming human lysozyme variant (I59T) using CEST and CPMG RD NMR at low pH. N CEST profiles probe the thermal unfolding of the native state into the denatured ensemble and reveal a distinct intermediate state. Global fitting of N CEST and CPMG data provides kinetic and thermodynamic parameters, characterizing the intermediate state populated at 0.6%. H CEST data further confirm the presence of the intermediate state displaying unusually high or low H chemical shifts. To investigate the structural details of this intermediate state, this work uses molecular dynamics (MD) simulations, which recapitulate the experimentally observed folding pathway and free energy landscape. This work observes a high-energy intermediate state with a locally disordered β-domain and C-helix, stabilized by non-native hydrogen bonding and amide-π interactions, accounting for its anomalous H chemical shifts. Together, these NMR and MD data provide the first direct structural information on the intermediate state, offering insights into targeting lysozyme amyloidosis.