Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements.
It is important to characterize drug-albumin binding during drug discovery and lead optimization as strong binding may reduce bioavailability and/or increase the drug's in vivo half-life. Despite knowing about the location of human serum albumin (HSA) drug binding sites and the residues important for binding, less is understood about the binding dynamics between exogenous drugs and endogenous fatty acids. In contrast to highly specific antibody-antigen interactions, the conformational flexibility of albumin allows the protein to adopt multiple conformations of approximately equal energy in order to accommodate a variety of ligands. Nuclear magnetic resonance (NMR) diffusion measurements are a simple way to quantitatively describe ligand-protein interactions without prior knowledge of the number of binding sites or the binding stoichiometry. This method can also provide information about ligand orientation at the binding site due to buildup of exchange-transferred NOE (trNOE) on the diffusion time scale of the experiment. The results of NMR diffusion and NOE experiments reveal multiple binding interactions of HSA with dansylglycine, a drug site II probe, and caprylate, a medium-chain fatty acid that also has primary affinity for HSA's drug site II. Interligand NOE (ilNOE) detected in the diffusion analysis of a protein solution containing both ligands provides insight into the conformations adopted by these ligands while bound in common HSA binding pockets. The results demonstrate the ability of NMR diffusion experiments to identify ternary complex formation and show the potential of this method for characterizing other biologically important ternary structures, such as enzyme-cofactor-inhibitor complexes.
Lucas
Laura H
LH
Department of Chemistry, University of Kansas, Lawrence, Kansas 66045, USA.
Price
Kristin E
KE
Larive
Cynthia K
CK
D001667:Binding, Competitive; D002210:Caprylates / Q000378:metabolism*; D004058:Diffusion; D018604:Epitope Mapping; D005998:Glycine / Q000031:analogs & derivatives* / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D012709:Serum Albumin / Q000276:immunology* / Q000378:metabolism*
D002210:Caprylates; D008024:Ligands; D012709:Serum Albumin; C038595:dansylglycine; D005998:Glycine