Molecular Basis for the Selectivity of DHA and EPA in Sudlow's Drug Binding Sites in Human Serum Albumin with the Combined Use of NMR and Docking Calculations.

Medium- and long-chain saturated and unsaturated free fatty acids (FFAs) are known to bind to human serum albumin (HSA), the main plasma carrier protein. Atomic-level structural data regarding the binding mode in Sudlow's sites I (FA7) and II (FA4, FA3) of the polyunsaturated ω-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), however, are largely unknown. Herein, we report the combined use of saturation transfer difference (STD) and Interligand NOEs for Pharmacophore Mapping (INPHARMA) NMR techniques and molecular docking calculations to investigate the binding mode of DHA and EPA in Sudlow's sites Ι and ΙΙ of HSA. The docking calculations and the significant number of interligand NOEs between DHA and EPA and the drugs warfarin and ibuprofen, which are stereotypical ligands for Sudlow's sites I and II, respectively, were interpreted in terms of competitive binding modes and the presence of two orientations of DHA and EPA at the binding sites FA7 and FA4. The exceptional flexibility of the long-chain DHA and EPA and the formation of strongly folded structural motives are the key properties of HSA-PUFA complexes.

NMR and computational studies reveal novel aspects in molecular recognition of unsaturated fatty acids with non-labelled serum albumin.

An approach based on the combined use of saturation transfer difference (STD), Tr-NOESY and Inter-ligand NOEs for PHArmacophore Mapping (INPHARMA) NMR techniques and docking calculations is reported, for the first time, for mapping interactions and specific binding sites of caproleic acid (10 : 1 cis-9), oleic acid (18 : 1 cis-9), linoleic acid (18 : 2 cis-9,12) and linolenic (18 : 3, cis-9,12,15) free fatty acids (FFAs) with non-labelled serum albumin (BSA/HSA). Significant negative inter-ligand NOEs between the FFAs and the drugs ibuprofen and warfarin, through competition experiments, were observed. The inter-ligand NOEs and docking calculations were interpreted in terms of competitive binding mode, the significant folding of the bis allylic region and the presence of two orientations of the FFAs in the warfarin binding site (FA7), due to two potential distinctive anchoring polar groups of amino acids. This conformational flexibility is the reason that, the location and conformational states of the FFAs in the binding site of warfarin could not be determined accurately, despite numerous available X-ray structural studies. α-Linolenic acid competes favourably with warfarin at the binding site FA7. Isothermal titration calorimetry experiments of the preformed HSA/α-linolenic acid complex upon titration with warfarin show a significant reduction in the binding constant of warfarin, in very good agreement with NMR and computational data. The combined use, therefore, of STD, Tr-NOESY and INPHARMA NMR, ITC and docking calculations may find promising applications in the field of protein-lipid recognition research.

Serum albumin as a primary non-covalent binding protein for nitro-oleic acid.

This work explores the interaction of 9/10-nitro-oleic acid (NO2-OA) with human serum albumin (HSA). The molecular mechanism of the biological action of NO2-OA is to our knowledge based on a reversible covalent reaction-Michael addition of nucleophilic amino acid residues of proteins. Since HSA is an important fatty acid transporter, a key question is whether NO2-OA can bind covalently or non-covalently to HSA, similarly to oleic acid (OA), which can interact with the FA1-FA7 binding sites of the HSA molecule. 1H NMR studies and competition analysis with OA and the drugs ibuprofen and warfarin were used to investigate a potential non-covalent binding mode. NO2-OA/HSA binding was confirmed to compete with warfarin for FA-7 with significantly higher affinity. NO2-OA competes with ibuprofen for FA-3 and FA-6, however, in contrast to the situation with warfarin, the binding affinities are not significantly different. The described interactions are based exclusively on non-covalent binding. No covalent binding of NO2-OA to HSA was detected by MS/MS. More detailed studies based on MALDI-TOF-MS and Ellman's assay indicated that HSA can be covalently modified in the presence of NO2-OA to a very limited extent. It was also shown that NO2-OA has a higher affinity to HSA than that of OA.

DFT Calculations of 1H- and 13C-NMR Chemical Shifts of Geometric Isomers of Conjugated Linoleic Acid (18:2 ω-7) and Model Compounds in Solution.

A density functional theory (DFT) study of the 1H- and 13C-NMR chemical shifts of the geometric isomers of 18:2 ω-7 conjugated linoleic acid (CLA) and nine model compounds is presented, using five functionals and two basis sets. The results are compared with available experimental data from solution high resolution nuclear magnetic resonance (NMR). The experimental 1H chemical shifts exhibit highly diagnostic resonances due to the olefinic protons of the conjugated double bonds. The "inside" olefinic protons of the conjugated double bonds are deshielded than those of the "outside" protons. Furthermore, in the cis/trans isomers, the signals of the cis bonds are more deshielded than those of the trans bonds. These regularities of the experimental 1H chemical shifts of the olefinic protons of the conjugated double bonds are reproduced very accurately for the lowest energy DFT optimized single conformer, for all functionals and basis sets used. The other low energy conformers have negligible effects on the computational 1H-NMR chemical shifts. We conclude that proton NMR chemical shifts are more discriminating than carbon, and DFT calculations can provide a valuable tool for (i) the accurate prediction of 1H-NMR chemical shifts even with less demanding functionals and basis sets; (ii) the unequivocal identification of geometric isomerism of CLAs that occur in nature, and (iii) to derive high resolution structures in solution.

High Resolution NMR Spectroscopy as a Structural and Analytical Tool for Unsaturated Lipids in Solution.

Mono- and polyunsaturated lipids are widely distributed in Nature, and are structurally and functionally a diverse class of molecules with a variety of physicochemical, biological, medicinal and nutritional properties. High resolution NMR spectroscopic techniques including 1H-, 13C- and 31P-NMR have been successfully employed as a structural and analytical tool for unsaturated lipids. The objective of this review article is to provide: (i) an overview of the critical 1H-, 13C- and 31P-NMR parameters for structural and analytical investigations; (ii) an overview of various 1D and 2D NMR techniques that have been used for resonance assignments; (iii) selected analytical and structural studies with emphasis in the identification of major and minor unsaturated fatty acids in complex lipid extracts without the need for the isolation of the individual components; (iv) selected investigations of oxidation products of lipids; (v) applications in the emerging field of lipidomics; (vi) studies of protein-lipid interactions at a molecular level; (vii) practical considerations and (viii) an overview of future developments in the field.