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 high-temperature molecular dynamics simulations, harmonic linear dis- criminant analysis (HLDA), and parallel-tempering metadynamics (PT-MetaD) to dis- sect the earliest conformational transitions of the disease-relevant SAA ₁₋₇₆ fragment. By constructing an optimized one-dimensional collective variable (sHLDA) from inter- helix contacts and helical root-mean-square deviations, we perform nearly 4 µ s of en- hanced sampling across 79 replicas (300–450 K). Free-energy surfaces projected onto sHLDA and radius of gyration reveal a possible misfolding trajectory comprising thir- teen metastable intermediates. We identify that helix III destabilizes first, preceding loss of helix II and helix I, while global compactness is retained. This stepwise mech- anism underscores the key role of helix III in the establishment of the native fold of SAA. Solvent-accessible surface-area analyses pinpoint a transient surge in exposure of the aggregation-prone core (residues 42–48) within specific misfolded intermediates, implicating localized core exposure rather than wholesale unfolding as the trigger for misfolding. Temperature-dependent secondary-structure profiling further confirms that SAA ₁₋₇₆ behaves as a folded bundle with disordered loops, rather than a fully disor- dered protein. These insights identify helix III stabilization and masking of the amy- loidogenic segment as strategic avenues for therapeutic intervention.