Localizing Fatty Acid Binding Sites on Human Serum Albumin by 2D-NMR

Abstract

The most abundant protein in plasma, human serum albumin (HSA), is the principal carrier for endogenous lipophilic compounds and lipophilic drugs. Non-esterified long chain fatty acids (FA) are its primary physiological ligand, with multiple binding sites of varying affinities. Detailed structural understandings of HSA-ligand interactions are vital during the development of drugs. HSA ligand interactions have been studied by various biophysical methods: x-ray crystallography, fluorescence spectroscopy, and nuclear magnetic resonance (NMR). Numerous crystal structures of HSA, with and without drugs and/or FAs, have been published. Compared to crystallography, our solution state NMR studies are focused on the more physiologically relevant state. Previously, we identified three high affinity and several low affinity FA binding sites on HSA by 1D-NMR at low resolution. Now, we are correlating crystallographic data with high-resolution 2D-NMR spectra of 13C-methyl-labeled oleic acid (OA). Our 2D-HSQC spectra show the 13C-methyl group of OA bound to HSA. The unique microenvironments that each methyl group experiences in the binding pockets gives rise to different NMR signals of varying intensities. These correspond to the different affinities that HSA has for OA, and our 2D-NMR spectra clearly differentiate the three highest affinity-binding sites from six lower affinity-binding sites. We have identified Sudlow's Drug Sites by utilizing the known FA competitors and Sudlow's site binders: warfarin and diazepam. By expanding this drug competition strategy with ibuprofen and diflunisal, we have identified additional FA binding sites. The highest affinity FA binding sites do not have known competitors and these sites are identified with the use of site-directed mutagenesis of HSA. In summary, we are reporting an approach for studying the interactions of FAs with HSA and FA-competitive drugs in a site-specific manner through the use of state-of-the-art 2D-NMR techniques.