Correspondence of Fatty Acid and Drug Binding Sites on Human Serum Albumin: A Two-Dimensional Nuclear Magnetic Resonance Study

Abstract

The ability of human serum albumin (HSA) to bind fatty acids (FA) in multiple sites has been revealed by many studies. Here we detect and characterize nine individual binding sites by two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy of 18-[(13)C]-oleic acid (OA) complexed with HSA. We characterize site-specific FA binding by addition of (i) different FA molar ratios (from 1:1 to 4:1 OA:HSA) to observe the order of filling and occupancy of binding sites; (ii) methyl-β-cyclodextrin, as a FA acceptor, to observe the dissociation of FA; and (iii) drugs (with known binding sites in the crystal structure) to reveal the correspondence of three NMR peaks with sites in the crystal structure. At 1:1 and 2:1 OA:HSA ratios, three sites were shown to fill sequentially. These high-affinity sites were well resolved from additional sites (one medium-affinity and five low-affinity) observed at 3:1 and 4:1 OA:HSA ratios. Methyl-β-cyclodextrin extracted OA from individual sites in the reverse order of filling. FA bound in three low-affinity sites were displaced by drugs shown to bind in crystalline HSA to FA sites 7 and 3 (Sudlow's drug sites I and II, respectively) and FA site 6. With this strategy, 2D NMR spectral analysis permits site-specific characterization of the binding of drugs and FA and provides a sensitive probe of the mutual effects of FA and ligand binding.

Probing the binding site characteristics of HSA: A combined molecular dynamics and cheminformatics investigation

“…The findings also support more recent NMR displacement studies which showed that FA bound to the low affinity FA6 and FA7 sites were easily displaceable [6]. Our results suggest that the more facile substrate displacement observed from sites FA6 and FA7 is a result of their greater solvent exposure (water contacts = 7.75 at FA6 and 8.43 at FA7) and fewer protein-FA hydrogen bonds and supports the findings from recent spectroscopic experiments [19].…”

“…To date approximately 100 HSA structures have been published in the Protein Data Bank (www.rcsb.org). In addition, NMR [5,6], EPR [7] and biochemical * Corresponding author. Tel.…”

CD36 Binds Oxidized Low Density Lipoprotein (LDL) in a Mechanism Dependent upon Fatty Acid Binding

“…We found that HSA bound and released OA rapidly and with a high RU response (for example, Fig. 1E), which was expected from extensive previous studies of FA binding, including those in our lab with albumin (39,40) and FABP1 (41,42). As an additional negative control, we bound IgG to the chip surface and saw no OA binding, as expected (Fig.…”

“…Nine individual sites on HSA have been recently been detected by two-dimensional NMR, and several NMR peaks were correlated with crystallographic sites well characterized for long chain FA (39). Binding constants measured by two-dimensional NMR on a site-specific basis varied only by a factor of 10 between the highest and lowest affinity sites, with affinity constant (K D ) values similar to those reported by earlier studies (note:…”

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

“…The binding property (e.g., binding sites, binding affinity or strength) of these molecules to serum albumins forms an important topic in pharmacokinetics. [1][2][3][4] Several analytical methods have been developed to study the binding properties of different drugs to serum albumins, such as X-ray crystallography, 5,6 nuclear magnetic resonance (NMR), [7][8][9][10][11] and surface plasmon resonance (SPR), 12,13 etc. However, these methods are constrained by either a tedious and time-consuming analyzing process (e.g., growth of single crystal for X-ray crystallographic study), requirement of specialized and expensive equipment (SPR), or in need of costly isotope labeling (NMR) for detection.…”