Mechanistic insights into dimerization and cross-β sheet formation in the HIV-associated amyloidogenic peptide PAP248-286 from unbiased all-atom molecular dynamics simulations.

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 indicate that peptide association and β-sheet formation are governed by a cooperative interplay between hydrogen bonding and hydrophobic interactions. 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.