A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin

• Accurate estimation of free testosterone (fT) is used widely in the diagnosis of hyogonadism.
• Complexity of direct measurements have led to development of models for calculation of fT.
• Current models used for fT estimation show systematic deviation from values measured by equilibrium dialysis.
• The SHBG exhibits complex behaviour in associating with testosterone.
• fT values from new mechanistic model incorporating allostery in SHBG:T match closely with those measured by equilibrium dialysis.

PurposeCirculating free testosterone (FT) levels have been used widely in the diagnosis and treatment of hypogonadism in men. Due to experimental complexities in FT measurements, the Endocrine Society has recommended the use of calculated FT (cFT) as an appropriate approach for estimating FT. We show here that the prevailing model of testosterone's binding to SHBG, which assumes that each SHBG dimer binds two testosterone molecules and that the two binding sites on SHBG have similar binding affinity is erroneous and provides FT values that differ substantially from those obtained using equilibrium dialysis.MethodsWe characterized testosterone's binding to SHBG using binding isotherms, ligand depletion curves, and isothermal titration calorimetry (ITC). We derived a new model of testosterone's binding to SHBG from these experimental data and used this model to determine FT concentrations and compare these values with those derived from equilibrium dialysis.ResultsExperimental data on testosterone's association with SHBG generated using binding isotherms including equilibrium binding, ligand depletion experiments, and ITC provide evidence of a multi-step dynamic process, encompassing at least two inter-converting microstates in unliganded SHBG, readjustment of equilibria between unliganded states upon binding of the first ligand molecule, and allosteric interaction between two binding sites of SHBG dimer. FT concentrations in men determined using the new multistep dynamic model with complex allostery did not differ from those measured using equilibrium dialysis. Systematic error in calculated FT vales in females using Vermeulen's model was also significantly reduced. In European Male Aging Study, the men deemed to have low FT (<2.5th percentile) by the new model were at increased risk of sexual symptoms and elevated LH.ConclusionTestosterone's binding to SHBG is a multi-step dynamic process that involves complex allostery within SHBG dimer. FT values obtained using the new model have close correspondence with those measured using equilibrium dialysis.