Effects of phosphorylation on the structure and assembly of Aβ, and the interaction with the receptor of advanced glycation end products (RAGE)

Abstract

Amyloid-β (Aβ) aggregation is a central event in Alzheimer's disease (AD), and serine phosphorylation is an important post-translational modification that affects both Aβ self-assembly and its interaction with binding partners. Here, we combined all-atom molecular dynamics (MD) simulations with biochemical and biophysical experiments to investigate the effects of site-specific phosphorylation on Aβ1–42 aggregation and its interaction with the receptor for advanced glycation end products (RAGE). Three Aβ1–42 variants were analyzed: non-phosphorylated Aβ1–42 (npAβ1–42), Ser8-phosphorylated Aβ1–42 (pS8Aβ1–42), and Ser26-phosphorylated Aβ1–42 (pS26Aβ1–42). Phosphorylation altered monomer conformations, intramolecular interactions, and surface electrostatics. In oligomeric systems, it weakened interpeptide interactions, reduced interchain β-sheet formation, and reshaped the free energy landscape, with pS26Aβ1–42 showing the greatest conformational heterogeneity and lowest stability during random assembly. These destabilizing effects were partly alleviated during stepwise assembly, which promoted more ordered β-structure formation and enhanced oligomer stability. Binding assays further showed that pS26Aβ bound soluble RAGE (sRAGE), endogenous secretory RAGE (esRAGE), and full-length RAGE (flRAGE) with higher affinity than npAβ and pS8Aβ. MD simulations and binding free energy calculations indicated that Ser26 phosphorylation stabilizes the Aβ–RAGE complex through enhanced hydrogen bonds, salt bridges, and hydrophobic interactions, with R29, H217, and R218 identified as key RAGE residues. In addition, sRAGE inhibited Aβ1–42 aggregation in a concentration-dependent manner, mainly by suppressing secondary nucleation. Together, these findings show that serine phosphorylation modulates Aβ1–42 aggregation and RAGE binding in a site-specific manner, providing mechanistic insights into Aβ pathology in AD.