Destabilization of Helix III Initiates Early Serum Amyloid A Misfolding by Exposing Its Amyloidogenic Core

Serum amyloid A (SAA) is the principal precursor of AA amyloidosis, yet the early molecular steps that trigger its pathological misfolding remain unclear. Here, we combine harmonic linear discriminant analysis (HLDA) and parallel-tempering metadynamics (PT-MetaD) to dissect the earliest conformational transitions of the disease-relevant SAA1–76 fragment. By constructing an optimized one-dimensional collective variable (sHLDA) from interhelix contacts and helical root-mean-square deviations, we perform 4 μs of enhanced sampling across 79 replicas (300–450 K). Free-energy surfaces reveal a misfolding trajectory where helix III destabilizes first, preceding loss of helices II and I while global compactness persists. Solvent-accessible surface-area analysis reveals transient exposure of the aggregation-prone core (residues 42–48) within specific intermediates, implicating localized core exposure rather than wholesale unfolding as the trigger for misfolding. Temperature-dependent secondary-structure profiling confirms that SAA1–76 behaves as a folded bundle with disordered loops. These findings highlight helix III stabilization and amyloidogenic segment masking as potential therapeutic strategies.