title: A multi-step, dynamic allosteric model of testosterone's binding to sex hormone binding globulin.
abstract: OBJECTIVE
  Circulating 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.
  
  
  METHODS
  We 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.
  
  
  RESULTS
  Experimental 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.
  
  
  CONCLUSIONS
  Testosterone'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.
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Mikhail N
authors.initials: MN
authors.lastname: Zakharov
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118, USA. Electronic address: sbhasin@partners.org.
authors.forename: Shalender
authors.initials: S
authors.lastname: Bhasin
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Travison
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Xue
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118, USA.
authors.forename: Jagadish
authors.initials: J
authors.lastname: Ulloor
authors.affiliation: Sections of Preventative Medicine and Cardiology, Boston University School of Medicine, 761 Harrison Court, Boston, MA 02118, USA.
authors.forename: Ramachandran S
authors.initials: RS
authors.lastname: Vasan
authors.affiliation: Andrology Research Unit, Manchester Academic Health Science Centre, Manchester Royal Infirmary, The University of Manchester, Manchester, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Carter
authors.affiliation: Andrology Research Unit, Manchester Academic Health Science Centre, Manchester Royal Infirmary, The University of Manchester, Manchester, UK.
authors.forename: Frederick
authors.initials: F
authors.lastname: Wu
authors.affiliation: Section of Endocrinology, Diabetes, and Nutrition, Boston Medical Center, Boston University School of Medicine, Boston, MA 02118, USA. Electronic address: jasuja1@gmail.com.
authors.forename: Ravi
authors.initials: R
authors.lastname: Jasuja
mesh_terms: D000494:Allosteric Regulation / Q000502:physiology; D005260:Female; D006801:Humans; D008297:Male; D008956:Models, Chemical; D055503:Protein Multimerization; D012738:Sex Hormone-Binding Globulin / Q000737:chemistry* / Q000378:metabolism; D013739:Testosterone / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012738:Sex Hormone-Binding Globulin; D013739:Testosterone
keywords: Allostery; Free testosterone; SHBG; Steroid transport; Testosterone