Research On The Interaction Between Perfluoroalkyl Substances And HL-FABP

Perfluoroalkyl substances (PFAAs) are a kind of organic pollutant with the similar structure of fatty acid that has highly persistent and bioaccumulative. PFAAs have been widely used in civil and industrial products resulting in the broad distribution in environmental samples, biological samples, and human bodies. Some indirect evidences indicate that liver is an important target organ for PFAAs, many proteins in surem or hepatic cells such as liver fatty acid binding acid (L-FABP) can interact with PFAAs, while the mechanism of these interactions remain poorly understood. L-FABP is abundant in the cytoplasm of liver cells and plays an important role in the transport and metabolism of fatty acids, moreover, its expression is under control of nuclear receptor including peroxisome proliferators-activated receptor (PPARa). In this study, we cloned and expressed hL-FABPand its four variants(S39G, M74G, N111D, R122G) and used CD assay, trypsin limited digestion, fluorescence displacement, isothermal titration calorimetry (ITC) assay and Molecular docking to research the interaction between PFAAs and hL-FABP in order to give a description of this interaction. The results are as follows:1 From fluorescence displacement and ITC assay, we confirmed that PFAAs (PFNA, PFOA, PFHxS, PFHxA) can bind with hL-FABP non-covalently by hydrophobic and hydrogen-bonding interaction. The affinity increased significantly with their carbon numbers. The affinity for four kinds of PFAAs binding to WT hL-FABP is in the order of PFNA>PFOA>PFHxS>PFHxA. The ITC assay reveals that PFOA/PFNA displayed a moderate affinity for hL-FABP binding sites at a 1:1 molar ratio.2 For the four variants, R122G and N111D changed significantly on molar ratio and binding affinity respectively:the molar ratio for R122G changed to 0.5:1; the affinity for N111D decreased obviously, while S39G and M74G have no significant difference compared to WT hL-FABP. CD assay, trypsin limited digestion and molecular docking assay shows that when binding with PFAAs the constructional changes of hL-FABP get more significant with PFAAs carbon number, which performed as obvirously increased a-helix cotents and decreased P-sheets. After substituting R122 with Gly, the original structure changed a lot (the volume of outter binding site enlarged) while the constructional changes caused by PFAAs were not observed, the reason may be that the constructional change mentioned above make PFAA access into inner binding site without "gate keeper" residues rotation.3 molecular docking results indicate that the process of PFAAs binding to hL-FABP can be describes as followed:under the orientation of N111, the first molecular of PFAA access to the outer binding site in a "head-in" mode, and can be pulled into inner site by R122 with a rotation at δ CH2, then the unoccupied outer binding site can bind with the second PFAA. The mode for PFAAs binding to hL-FABP is similar to long chain fatty acid that can bind with both two binding sites. In this process, R122 and N111 play a crucial role.