Broad PFAS Binding with Fatty Acid Binding Protein 4 Is Enabled by Variable Binding Modes

Abstract

Per- and polyfluoroalkyl substances (PFAS) are ubiquitous pollutants that bioaccumulate in wildlife and humans, yet the molecular basis of their protein interactions remains poorly understood. Here, we show that human adipocyte fatty acid-binding protein 4 (FABP4) can bind a diverse array of PFAS, including next-generation replacements for legacy chemicals and longer-chain perfluorocarboxylic acids. Shorter-chain PFAS, although weaker binders, still displayed measurable affinities, surpassing those of their nonfluorinated analogs. We determined crystal structures of FABP4 bound to perfluorooctanoic acid (PFOA), perfluorodecanoic acid (PFDA), and perfluorohexadecanoic acid (PFHxDA), revealing three distinct binding modes. Notably, PFOA binds in two separate sites, and two distinct conformations define single-ligand binding of PFDA and PFHxDA. These arrangements enhance hydrophobic interactions within the binding cavity and likely explain the low micromolar dissociation constants observed in fluorescence competition assays. Our findings underscore the critical roles of chain length, headgroup functionality, and protein conformation in PFAS-FABP4 interactions. Given the emerging implications of the role of FABP4 in endocrine function, even subtle PFAS-induced perturbations could affect metabolic regulation and disease risk. Overall, this work highlights the value of direct structural and biochemical insights into PFAS-FABP4 interactions and paves the way for future research on PFAS transport and toxicological outcomes.

Keywords: FABP4; Fatty acid binding protein 4; PFAS; X-ray crystallography; fluorinated ligands; per- and polyfluoroalkyl substances; protein structure.

1

Structures and FABP4 binding affinities of compounds examined in this study. (a) Chemical structures of PFAS and alkanoic acid compounds that were measured for affinity to FABP4. (b) Binding affinity (K_d) of perfluorocarboxylic acids compounds and their alkanoic acid analogs with FABP4. (c) Affinity of PFAS compounds analogous to perfluorononanoic acid (PFNA) with alternate headgroups to FABP4. K_d values were converted from IC₅₀ measurements obtained from ANS displacement assays. Error bars are smaller than the diameter of the symbols. See Table S2 for detailed values including errors.

2

Crystal structures of PFOA, PFDA, and PFHxDA bound to FAPB4. (a) X-ray crystallographic structure of PFOA (spheres) bound to FABP4 (magenta cartoon), displaying two PFOA molecules bound. (PDB: 9MIW). (b) X-ray crystallographic structure of FABP4 (wheat cartoon) with a single PFDA bound (PDB: 9MIZ). (c) X-ray crystallographic structure of FABP4 (cyan cartoon) with a single PFHxDA bound (PDB: 9MP2). Zoomed images of the (d) “primary” PFOA, (e) PFDA, or (f) PFHxDA bound to the hydrophobic ligand binding cavity. The protein cavity is depicted as a semitransparent surface, colored according to atomic identity: gray for hydrogens, blue for nitrogens, and red for oxygens, with carbons colored according to the cartoon coloring scheme. PFOA, PFNA, and PFHxDA are depicted in sticks, with key hydrogen-bonds and charge interactions highlighted in dashed yellow lines. For clarity, the water mediated H-bond between PFDA and S53 is omitted in (e). Resolved water molecules that mediate PFAS–protein interactions are depicted in red spheres.

3

Phe57 changes conformation to accommodate PFAS ligands. Zoom of the region surrounding Phe57 in the crystal structures of (a) apo-FABP4 (PDB: 9OB7), (b) PFOA-bound FABP4 (PDB: 9MIW), (c) PFDA-bound FABP4 (PDB: 9MIZ), and (d) PFHxDA-bound FABP4 (PDB: 9MP2). Phe57 and the PFAS ligands are displayed as sticks and the ligand binding cavity is displayed as a semitransparent surface.