No abstract

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Human serum albumin (HSA) acts as a carrier for testosterone, other sex hormones, fatty acids, and drugs. However, the dynamics of testosterone's binding to HSA and the structure of its binding sites remain incompletely understood. Here, we characterized the dynamics of testosterone's binding to HSA and the stoichiometry and structural location of the binding sites using two-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, bis-ANS partitioning, and equilibrium dialysis, complemented by molecular modeling. 2D NMR studies showed that testosterone competitively displaced 18-[ 13C]-oleic acid from at least three known fatty acid binding sites on HSA that also bind many drugs. Binding isotherms of testosterone's binding to HSA generated using fluorescence spectroscopy and equilibrium dialysis were nonlinear and the apparent Kd varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to two independent binding sites; the binding isotherms were most consistent with two or more allosterically coupled binding sites. Molecular dynamics studies revealed that testosterone's binding to fatty acid binding site 3 on HSA was associated with conformational changes at site 6, indicating that residues in in these two distinct binding sites are allosterically coupled. Conclusions There are multiple, allosterically coupled binding sites for testosterone on HSA. Testosterone shares these binding sites on HSA with free fatty acids, which could displace testosterone from HSA under various physiological states or disease conditions, affecting its bioavailability.

Summary Sex-hormone-binding globulin (SHBG) regulates the transport and bioavailability of estradiol. The dynamics of estradiol's binding to SHBG are incompletely understood, although it is believed that estradiol binds to each monomer of SHBG dimer with identical affinity (K d ∼2 nM). Contrary to the prevalent view, we show that estradiol's binding to SHBG is nonlinear, and the "apparent" K d changes with varying estradiol and SHBG concentrations. Estradiol's binding to each SHBG monomer influences residues in the ligand-binding pocket of both monomers and differentially alters the conformational and energy landscapes of both monomers. Monomers are not energetically or conformationally equivalent even in fully bound state. Estradiol's binding to SHBG involves bidirectional, inter-monomeric allostery that changes the distribution of both monomers among various energy and conformational states. Inter-monomeric allostery offers a mechanism to extend the binding range of SHBG and regulate hormone bioavailability as estradiol concentrations vary widely during life.