Unveiling the structural insights of PFAS-β-lactoglobulin binding mechanism mediating neuronal toxicity in neonates.

The strong electrostatic interaction in C-F bonds contributes to the forever nature of per- and polyfluoroalkyl (PFAS) substances. The frequent use of PFAS in consumer products results in diverse toxic effects. Previous studies present its ability to cross the blood-brain and placental barriers and its presence in breast milk and infant formula, causing severe neurotoxic effects. Beta-lactoglobulin, a milk protein, transports hydrophobic and amphiphilic compounds to different organs, including the brain. Biochemical and biophysical studies of beta-lactoglobulin-PFAS interaction have been promising. The present study reports the complex crystal structures of beta-lactoglobulin with PFOA (Perfluorooctanoic acid) at 2.0 Angstrom, PFOS (Perfluorooctanesulfonic acid) at 2.5 Angstrom, and PFDA (Perfluorodecanoic acid) at 2.0 Angstrom, disclosing the high affinity of the three PFAS compounds for the central calyx of beta-lactoglobulin. Significant hydrophobic interactions stabilized the binding of the PFAS hydrophobic tail inside the calyx, while polar interactions with residues Lys60 and Lys69 stabilized the polar head group. Comparative structural analysis revealed the presence of an open conformation of the EF loop containing the Glu89 latch residue in the complex structures compared to the apo-form. Molecular dynamics (MD) simulation analysis revealed high stability of the PFAS binding and attainment of global minima in all complexes. The average binding energy of PFDA in beta-lactoglobulin calyx was -25.04 kcal/mol, which was higher than PFOS (-21.47 kcal/mol) and PFOA (-22.89 kcal/mol) due to increased van der Waals interactions of the longer hydrophobic chain of PFDA with beta-lactoglobulin. Henceforth, this work presents the structural mechanism of PFAS-beta-lactoglobulin binding, disclosing beta-lactoglobulin as an effective PFAS transporter mediating neurotoxicity in neonates.