PubMed · 474746
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D Rodbard. 1979. Negative cooperativity: a positive finding?. https://doi.org/10.1152/ajpendo.1979.237.3.e203
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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.
Prolactin, a lactogenic hormone, binds to two prolactin receptors sequentially, the first receptor binding at site 1 of the hormone followed by the second receptor binding at site 2. We have investigated the mechanism by which human prolactin (hPRL) binds the extracellular domain of the human prolactin receptor (hPRLbp) using surface plasmon resonance (SPR) technology. We have covalently coupled hPRL to the SPR chip surface via coupling chemistries that reside in and block either site 1 or site 2. Equilibrium binding experiments using saturating hPRLbp concentrations show that site 2 receptor binding is dependent on site 1 receptor occupancy. In contrast, site 1 binding is independent of site 2 occupancy. Thus, sites 1 and 2 are functionally coupled, site 1 binding inducing the functional organization of site 2. Site 2 of hPRL does not have a measurable binding affinity prior to hPRLbp binding at site 1. After site 1 receptor binding, site 2 affinity is increased to values approaching that of site 1. Corruption of either site 1 or site 2 by mutagenesis is consistent with a functional coupling of sites 1 and 2. Fluorescence resonance energy transfer (FRET) experiments indicate that receptor binding at site 1 induces a conformation change in the hormone. These data support an "induced-fit" model for prolactin receptor binding where binding of the first receptor to hPRL induces a conformation change in the hormone creating the second receptor-binding site.