β-lactoglobulin–per- and polyfluoroalkyl substance-binding interactions identifies the calyx domain as a determinant of contaminated milk exposure and the calycin protein family as potential mediators of per- and polyfluoroalkyl substance toxicity

Per- and polyfluoroalkyl substances (PFAS) are a diverse class of highly fluorinated persistent synthetic chemical pollutants. Major routes of human exposure include ingestion of contaminated drinking water and foods including dairy. Consumption of PFAS-contaminated milk and dairy is especially concerning for infants and children who are particularly sensitive and most highly exposed. Here, we report findings of quantitative analysis of PFAS binding to β-lactoglobulin (β-Lg), the major whey protein in bovine milk, using differential scanning fluorimetry to determine binding affinities for 17 PFAS; except for uncharged fluorotelomer alcohols, β-Lg bound each PFAS congener tested, supporting a key role of charged functional groups in binding. The perfluoroalkyl carboxylic acid trifluoroacetic acid (TFA) bound with lowest affinity (Kd=8.6 mM) and long-chain congeners PFNA, PFDA, and PFUnDA bound with highest affinities. Evidence of significant cooperative binding was found for TFA, PFDA, PFUnDA, and PFOS. Molecular docking was used to define molecular mechanisms of PFAS binding by β-Lg and across the calycin super family of lipocalins and fatty acid-binding proteins. All calycins were predicted to bind PFAS in the calyx domain with ΔG of binding ranging from -5.3 to -9.4 kcal/mol, revealing that the binding affinity for many PFAS is greater than those for binding albumin. In total, this study has identified the calycin protein superfamily as PFAS-binding proteins, most of which have well-characterized functions related to key endocrine and toxicological pathways associated with the adverse consequences of PFAS exposure.