Mechanistic Insights into Cross-β Sheet Formation in the HIV-Associated Amyloidogenic Peptide PAP248–286 from Unbiased All-Atom Molecular Dynamics Simulation

Semen-derived enhancer of viral infection (SEVI) fibrils, assembled from the peptide fragment PAP248–286, enhance HIV transmission by promoting viral attachment to host cells. However, the molecular basis of SEVI nucleation and early aggregation remains unclear. Here, we conducted 80 independent all-atom molecular dynamics (MD) simulations spanning a total of 40 μs, together with 100 independent steered MD and umbrella sampling runs, to explore the dimerization and dissociation of PAP248–286 Our results show that hydrogen bonding is the dominant stabilizing force driving β-sheet formation during peptide association. Residue-level analyses identified Arg10, Val17, Glu19, and Ile20 as key contributors to inter-peptide binding, consistent with steric zipper motifs described in other amyloid systems. Steered MD revealed mechanically resilient dimers with average rupture forces of ∼20 kcal/mol/Å and multi-barrier unbinding behavior. Umbrella sampling estimated a peptide dissociation free energy of ∼8.7 kcal/mol, highlighting a clear thermodynamic separation between bound and unbound states. Together, these findings suggest that small β-sheet nuclei in PAP248–286 dimers act as cooperative intermediates that seed the formation of full-length cross-β structures, providing atomistic insights into the earliest steps of SEVI fibril assembly.