Allosterically Coupled Multisite Binding of Testosterone to Human Serum Albumin

Abstract

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.

Free testosterone and cardiometabolic parameters in men: comparison of algorithms

“…2 ). However, the algorithm by Zakharov et al tended to be in closer agreement with the associations seen for total testosterone indicating that the algorithm is more dependent on the levels of total testosterone compared to the algorithm by Vermeulen et al However, with findings from recent studies on crystal structure and multisite binding of testosterone with albumin ( 31 , 32 ), it is conceivable that simplified assumptions in extant models should be examined in detail. It is unclear why the algorithm by Vermulen et al was largely independent of both the levels of SHBG, albumin and total testosterone level in contrast to the algorithm by Zakharov.…”

Association of sex steroid hormones and new bone formation rate after iliac onlay grafting: a prospective clinical pilot study

“…As measurement of free sex steroid hormone concentration are complex and time-consuming, calculation of free sex steroid hormones, using measured total E2/total T and SHBG in conjunction with equilibrium-binding theory or empirical equations became popular, regardless of their unreliability [ 44 , 45 ]. Equilibrium-binding equation-based calculation methods presented to be even worse than assumption-free empirical formulae [ 44 , 46 ], as the SHBG as well as the albumin association constant for E2/T differs up to 14-fold between different authors [ 16 , 47 , 48 ]. Therefore, results based on the calculated free androgen index should be considered with caution.…”

Estradiol induces allosteric coupling and partitioning of sex-hormone-binding globulin monomers among conformational states

“…For instance, at a low partial pressure of oxygen, a relatively greater fraction of oxygen remains unbound to hemoglobin, whereas at higher partial pressures of oxygen, more oxygen becomes bound. We also have found evidence on allostery in testosterone's binding to its various binding sites on human serum albumin [ Jayaraj et al., 2021 ].…”