Phenylalanine Fibrils Seed Native Lysozyme to Form Membrane-Active Pathological Complexes at Physiological Conditions

Accumulation of metabolites at pathological concentrations can trigger nonclassical protein aggregation pathways that remain poorly understood. Here, we explore the molecular mechanism by which the metabolite phenylalanine (Phe), known to form amyloid-like fibrils in Phenylketonuria disease, induces a globular protein to form complex structures. We demonstrate that Phe fibrils act as molecular scaffolds to promote aggregation of a stable globular protein lysozyme (Lyz) under physiological conditions (pH 7.4, 37 °C), resulting in the formation of a heteronuclear Lyz-Phe complex. Spectroscopic and biophysical analyses reveal that π-π interactions between Trp residues of Lyz and the aromatic surface of Phe fibrils drive the initial association; additional hydrophobic interactions stabilize the complex. Unlike classical amyloidogenesis dominated by β-sheet enrichment in proteins, this heteronuclear complexation induces a substantial increment of random coil content in the protein's secondary structure with only a modest increase in the β-sheet content. The resulting complex strongly interacts with phospholipid bilayer membranes, compromises the membrane integrity, and exhibits significant cytotoxicity toward cultured mammalian fibroblast cells (L929). This study offers a hitherto unexplored alternative aggregation mechanism in which a metabolite imbalance, rather than intrinsic protein instability, converts a native globular protein into membrane-active, cytotoxic assemblies.