Endocrinology Journal Article

Allosterically Coupled Testosterone Binding to HSA

February 08, 2021
 

Abhilash Jayaraj, Heidi A Schwanz, Daniel J Spencer, Shalender Bhasin, James A Hamilton, B Jayaram, Anna L Goldman, Meenakshi Krishna, Maya Krishnan, Aashay Shah, Zhendong Jin, Eileen Krenzel, Sashi N Nair, Sid Ramesh, Wen Guo, Gerhard Wagner, Haribabu Arthanari, Liming Peng, Brian Lawney, Ravi Jasuja
Endocrinology, Volume 162, Issue 2, February 2021, bqaa199
https://doi.org/10.1210/endocr/bqaa199

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 characterize the dynamics of testosterone’s binding to HSA and the stoichiometry and structural location of the binding sites using 2-dimensional nuclear magnetic resonance (2D NMR), fluorescence spectroscopy, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt partitioning, and equilibrium dialysis, complemented by molecular modeling.

2D NMR studies showed that testosterone competitively displaced 18-[13C]-oleic acid from at least 3 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 dissociation constant varied with different concentrations of testosterone and HSA. The binding isotherms neither conformed to a linear binding model with 1:1 stoichiometry nor to 2 independent binding sites; the binding isotherms were most consistent with 2 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 2 distinct binding sites are allosterically coupled.

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.

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