madman
Super Moderator
Estradiol induces Allosteric Coupling and Partitioning of Sex Hormone Binding Globulin Monomers Among Conformational States
Ravi Jasuja, Daniel Spencer, Abhilash Jayaraj, Liming Peng, Meenakshi Krishna, Brian Lawney, Priyank Patel, B. Jayaram, Kelly M. Thayer, David L. Beveridge, Shalender Bhasin
Abstract
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 (Kd ~2 nM). Contrary to the prevalent view, we show that estradiol’s binding to SHBG is nonlinear and the "apparent" Kd 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 a fully-bound state.
Conclusion: 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.
INTRODUCTION
As living organisms became multicellular and more complex, hormones and circulating systems evolved to enable communication among distantly located cells and organs. The circulating binding proteins facilitated the transport of hormones and nutrients to various target tissues in the body. In humans and most mammalian species, most hormones are transported in the circulation, bound to their cognate binding proteins, and that their bioavailability to the target tissues and their biological activity is regulated by the circulating concentration of the non-protein bound fraction or the "free" hormone. The concept of the important role of binding proteins in regulating the bioavailability and biological activity of their ligands applies also to nutrients, such as vitamin D and B12, and many commonly used drugs, such as aspirin, warfarin, and some antibiotics.
Despite widespread adoption of the free hormone hypothesis, the dynamics of how hormones bind to their cognate binding proteins have remained incompletely understood. Among the various physiologic ligands, the binding of sex hormones, estradiol, and testosterone, to their high-affinity binding partner, sex hormone-binding globulin (SHBG), remains the most extensively studied. Estradiol (E2), the dominant estrogen in men and women, is found in human circulation bound primarily to sex hormone-binding globulin (SHBG) and human serum albumin (HSA) [Anderson, 1974; Dunn et al., 1981; Moll et al., 1981; Tietz, 1986; Peters, 1996; Pearlman et al., 1969; Burke and Anderson, 1972; Vigersky et al., 1979]. These circulating binding proteins regulate the transport, bioavailability, and metabolism of estradiol [Goldman et al.,2017; Rosner and Smith, 1975; Manni et al., 1985; Nisula and Dunn, 1979; Murphy, 1968; Zeginiadou et al., 1997; Laurent et al., 2016; Laurent and Vanderschueren, 2014]. Using an SHBG transgenic mouse model, Laurent et al. demonstrated that SHBG regulates the physiological function and the circulating half-life of sex steroids in vivo.
The dynamics of estradiol’s binding to SHBG remain incompletely understood. It is generally believed that estradiol binds with high affinity to a single binding pocket in each of the two monomers of the SHBG dimer [Grishkovskaya et al., 2000; Grishkovskaya et al., 1999; Avvakumov et al., 2001] and prior studies have reported a single Kd (~2 nM) for each monomer [Dunn et al., 1981; Moll et al., 1981; Burke and Anderson, 1972; Avvakumov et al., 2001; Sӧdergard et al., 1982; Vermeulen et al., 1999; Grishkovskaya et al., 2002b; Mazer, 2009]. Underlying these studies, however, is the assumption that estradiol’s binding to SHBG is linear and follows a one-to-one stoichiometry with an identical affinity for both monomers.
While the earlier studies assumed that there was an estradiol binding pocket at the interface of the SHBG dimer [Sui et al., 1996], subsequent resolution of the crystal structure of the N-terminal recombinant human SHBG containing the ligand-binding pocket (LBP) complexed with steroidal ligands [Grishkovskaya et al., 2000; Grishkovskaya et al., 1999; Avvakumov et al., 2001] revealed a homo-dimeric structure in which each monomer contains an LBP for estradiol. Observations that dimerization deficient SHBG variants bound estradiol with an affinity similar to that of wild-type SHBG [Avvakumov et al., 2001; Petra et al., 2001] led to the now-common view that the binding of estradiol to each monomer is equivalent and independent of its binding to the second monomer. Since then, the linear binding model with a Kd of ~2 nM for each monomer has remained the prevalent dogma in the literature.
Several published observations are inconsistent with the prevalent notion of linear binding of estradiol to SHBG in which both binding sites on the SHBG dimer are equivalent in their binding affinity. First, only a narrow range of estradiol concentrations was used in the binding data, which were fit to linear Scatchard plots to derive a single Kd. [Dunn et al., 1981; Moll et al., 1981; Burke and Anderson, 1972; Sӧdergard et al., 1982]. The linear transformation of data over a limited range of hormone concentrations may have prevented a complete understanding of estradiol association dynamics. Second, widely varying binding affinities have been reported for estradiol binding, ranging from as low as picomolar [Wu et al., 1976] to as high as 25 nM [Sui et al., 1996], depending on the estradiol and SHBG concentrations. These findings suggest that the apparent Kd might be affected by the estradiol concentrations and the estradiol to SHBG ratio, which would only be possible if there were a concentration-dependent allosteric interaction between the two binding sites on the SHBG dimer. Third, even in the presence of super-saturating estradiol concentrations, the crystal structure of the second monomer within the SHBG dimer could not be resolved. One possible explanation for the failure to resolve the crystal structure of the second monomer is that the two monomers are not equivalent in their conformations and energy states even though they have the same amino acid sequence [Grishkovskaya et al., 1999].
Although the estimates of estradiol’s binding affinity to SHBG have varied among studies, it is generally believed that its affinity is slightly lower than that of testosterone [Dunn et al., 1981; Moll et al., 1981; Burke and Anderson, 1972; Sӧdergard et al., 1982; Orwoll et al., 2006]. If these estimates of the relative binding affinities of estradiol and testosterone are correct, and if both bind to the same pocket in SHBG, then, given the much higher serum concentration of testosterone than estradiol in men (5000-6000 pg·mL-1 for testosterone versus 20-50 pg·mL-1 for estradiol), substantially less estradiol should be bound to SHBG under physiological conditions than what is observed [Dunn et al., 1981; Burke and Anderson, 1972; Sӧdergard et al., 1982]. Thus, it is difficult to reconcile these data with the notion of linear binding kinetics and a single Kd of ~2 nM.
To gain a better understanding of the binding dynamics of this system, we employed multiple biophysical techniques, modern computational tools, and Markov state modeling to examine nonlinear data derived from the binding isotherms and depletion curves. Some of the original studies were limited by the varying dialysis conditions, failure to account for protium-tritium exchange when using tritium-labeled tracers, and the inclusion of a narrow range of estradiol and SHBG concentrations. As described in the methods section, we took several steps to overcome these methodological concerns and to minimize their influence. Our studies provide evidence of a nonlinear, dynamic binding process involving allosteric coupling between the SHBG monomers that changes the energy landscape of both monomers and their distribution between various energy states such that the two monomers are not equivalent, even in the fully bound state and offer functional insight into the ligand-induced, intermonomeric interactions and conformational heterogeneity in SHBG.
3. RESULTS
3.1 Estradiol binding to SHBG exhibits complex interaction dynamics.
3.2 Intrinsic tryptophan emission from SHBG provides evidence that estradiol binding is multiphasic and associated with changes in the tryptophan microenvironment.
3.3 Estradiol-induced molecular rearrangement alters the conformational states of residues in the ligand-binding pockets of SHBG monomers, suggesting intermonomeric allosteric coupling.
3.4 Time-resolved lifetime fluorescence spectroscopy using bis-ANS demonstrates that estradiol binding significantly alters the global conformational state of the SHBG: E2 complex.
3.5 Dynamic cross-correlation matrix analysis shows allosteric changes in residue correlations upon estradiol binding to either of the two monomers.
3.6 Markov state models reveal dynamic allosteric conformational coupling between the SHBG monomers.
DISCUSSION
*The ligand-induced allosteric interaction between the monomers observed during estradiol's binding to SHBG may be a more general mechanism among multimeric binding proteins. We have previously found evidence of ensemble allostery in testosterone's binding to SHBG [Zakharov et al., 2015]. Our subsequent studies of testosterone's binding to human serum albumin revealed that testosterone can bind multiple (at least 6) binding sites on HSA and that testosterone binding to one site allosterically affects residues distant from the binding site (Jayaraj et al., 2020]. Variations in the apparent Kd depending on the relative concentrations of ligand and the binding protein also has been noted in studies of vitamin D binding to vitamin D binding protein. Similar dynamics in conformational ensemble and energetic repartitioning of protein populations have been shown to be functionally important in other physiologic systems, including the tetrameric hemoglobin, which exhibits intersubunit allostery in oxygen binding [Perutz, 1970; Monod et al., 1965; Koshland et al., 1966; Hilser et al., 2012; Motlagh et al., 2012].
Why would nature create such an allosteric mechanism in binding proteins?
Our findings of nonlinear dynamics of estradiol’s binding and the allosteric coupling of monomers within SHBG have potential physiological implications. First, the dynamic changes in Kd enabled by the dynamic conformational changes in SHBG upon ligand binding provide a versatile mechanism for extending the range of estradiol binding than would be possible if there were a single fixed Kd. The estradiol concentrations vary widely during different phases of the reproductive and nonreproductive phases of an individual’s life-extending from 1 to 6 pg/mL early in life and during menopause to ~ 30 to 500 pg/mL during different phases of the normal menstrual cycle to 30,000 to 40,000 pg/mL during pregnancy. Second, the nonlinear dynamics of estradiol’s binding and the allosteric coupling offer a potential mechanism for facile regulation of free hormone bioavailability under different physiologic and disease conditions. An example of this facile regulation is observed in men with hyperthyroidism some of whom develop gynecomastia. Hyperthyroidism is associated with increased levels of SHBG, and alterations in the relative ratio of free estradiol to free testosterone concentrations which have been implicated in the pathophysiology of breast enlargement in some hyperthyroid men [Chopra, 1974] Similar non-linear processes have been found in other biological systems; we speculate that this may be a more general mechanism in nature to regulate the bioavailability of nutrients and hormones. For instance, at a low partial pressure of oxygen, a relatively greater fraction of oxygen remains unbound to hemoglobin, while 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, 2021].
In conclusion, we show that estradiol binding to dimeric SHBG is a dynamic, nonlinear process that involves allosteric interaction between the two monomers of SHBG. The binding of estradiol to SHBG induces intra-molecular rearrangements in the estradiol binding pocket of the ligand-occupied monomer as well as in the binding pocket of the second monomer and alters the energy landscape of both monomers. The inter-monomeric allosteric communication is bidirectional – the binding of the second estradiol molecule also impacts the landscape and probability of conformational transitions in the first monomer, which was already bound to estradiol. Allosteric coupling in the SHBG monomers changes the energy landscape such that the two monomers are not equivalent even in the fully bound state. The allosteric interaction in the SHBG dimer may offer a potential mechanism to extend the dynamic binding range and to regulate the bioavailability of estradiol as the estradiol concentrations change several thousand-fold during the various phases of a person's life.
Ravi Jasuja, Daniel Spencer, Abhilash Jayaraj, Liming Peng, Meenakshi Krishna, Brian Lawney, Priyank Patel, B. Jayaram, Kelly M. Thayer, David L. Beveridge, Shalender Bhasin
Abstract
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 (Kd ~2 nM). Contrary to the prevalent view, we show that estradiol’s binding to SHBG is nonlinear and the "apparent" Kd 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 a fully-bound state.
Conclusion: 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.
INTRODUCTION
As living organisms became multicellular and more complex, hormones and circulating systems evolved to enable communication among distantly located cells and organs. The circulating binding proteins facilitated the transport of hormones and nutrients to various target tissues in the body. In humans and most mammalian species, most hormones are transported in the circulation, bound to their cognate binding proteins, and that their bioavailability to the target tissues and their biological activity is regulated by the circulating concentration of the non-protein bound fraction or the "free" hormone. The concept of the important role of binding proteins in regulating the bioavailability and biological activity of their ligands applies also to nutrients, such as vitamin D and B12, and many commonly used drugs, such as aspirin, warfarin, and some antibiotics.
Despite widespread adoption of the free hormone hypothesis, the dynamics of how hormones bind to their cognate binding proteins have remained incompletely understood. Among the various physiologic ligands, the binding of sex hormones, estradiol, and testosterone, to their high-affinity binding partner, sex hormone-binding globulin (SHBG), remains the most extensively studied. Estradiol (E2), the dominant estrogen in men and women, is found in human circulation bound primarily to sex hormone-binding globulin (SHBG) and human serum albumin (HSA) [Anderson, 1974; Dunn et al., 1981; Moll et al., 1981; Tietz, 1986; Peters, 1996; Pearlman et al., 1969; Burke and Anderson, 1972; Vigersky et al., 1979]. These circulating binding proteins regulate the transport, bioavailability, and metabolism of estradiol [Goldman et al.,2017; Rosner and Smith, 1975; Manni et al., 1985; Nisula and Dunn, 1979; Murphy, 1968; Zeginiadou et al., 1997; Laurent et al., 2016; Laurent and Vanderschueren, 2014]. Using an SHBG transgenic mouse model, Laurent et al. demonstrated that SHBG regulates the physiological function and the circulating half-life of sex steroids in vivo.
The dynamics of estradiol’s binding to SHBG remain incompletely understood. It is generally believed that estradiol binds with high affinity to a single binding pocket in each of the two monomers of the SHBG dimer [Grishkovskaya et al., 2000; Grishkovskaya et al., 1999; Avvakumov et al., 2001] and prior studies have reported a single Kd (~2 nM) for each monomer [Dunn et al., 1981; Moll et al., 1981; Burke and Anderson, 1972; Avvakumov et al., 2001; Sӧdergard et al., 1982; Vermeulen et al., 1999; Grishkovskaya et al., 2002b; Mazer, 2009]. Underlying these studies, however, is the assumption that estradiol’s binding to SHBG is linear and follows a one-to-one stoichiometry with an identical affinity for both monomers.
While the earlier studies assumed that there was an estradiol binding pocket at the interface of the SHBG dimer [Sui et al., 1996], subsequent resolution of the crystal structure of the N-terminal recombinant human SHBG containing the ligand-binding pocket (LBP) complexed with steroidal ligands [Grishkovskaya et al., 2000; Grishkovskaya et al., 1999; Avvakumov et al., 2001] revealed a homo-dimeric structure in which each monomer contains an LBP for estradiol. Observations that dimerization deficient SHBG variants bound estradiol with an affinity similar to that of wild-type SHBG [Avvakumov et al., 2001; Petra et al., 2001] led to the now-common view that the binding of estradiol to each monomer is equivalent and independent of its binding to the second monomer. Since then, the linear binding model with a Kd of ~2 nM for each monomer has remained the prevalent dogma in the literature.
Several published observations are inconsistent with the prevalent notion of linear binding of estradiol to SHBG in which both binding sites on the SHBG dimer are equivalent in their binding affinity. First, only a narrow range of estradiol concentrations was used in the binding data, which were fit to linear Scatchard plots to derive a single Kd. [Dunn et al., 1981; Moll et al., 1981; Burke and Anderson, 1972; Sӧdergard et al., 1982]. The linear transformation of data over a limited range of hormone concentrations may have prevented a complete understanding of estradiol association dynamics. Second, widely varying binding affinities have been reported for estradiol binding, ranging from as low as picomolar [Wu et al., 1976] to as high as 25 nM [Sui et al., 1996], depending on the estradiol and SHBG concentrations. These findings suggest that the apparent Kd might be affected by the estradiol concentrations and the estradiol to SHBG ratio, which would only be possible if there were a concentration-dependent allosteric interaction between the two binding sites on the SHBG dimer. Third, even in the presence of super-saturating estradiol concentrations, the crystal structure of the second monomer within the SHBG dimer could not be resolved. One possible explanation for the failure to resolve the crystal structure of the second monomer is that the two monomers are not equivalent in their conformations and energy states even though they have the same amino acid sequence [Grishkovskaya et al., 1999].
Although the estimates of estradiol’s binding affinity to SHBG have varied among studies, it is generally believed that its affinity is slightly lower than that of testosterone [Dunn et al., 1981; Moll et al., 1981; Burke and Anderson, 1972; Sӧdergard et al., 1982; Orwoll et al., 2006]. If these estimates of the relative binding affinities of estradiol and testosterone are correct, and if both bind to the same pocket in SHBG, then, given the much higher serum concentration of testosterone than estradiol in men (5000-6000 pg·mL-1 for testosterone versus 20-50 pg·mL-1 for estradiol), substantially less estradiol should be bound to SHBG under physiological conditions than what is observed [Dunn et al., 1981; Burke and Anderson, 1972; Sӧdergard et al., 1982]. Thus, it is difficult to reconcile these data with the notion of linear binding kinetics and a single Kd of ~2 nM.
To gain a better understanding of the binding dynamics of this system, we employed multiple biophysical techniques, modern computational tools, and Markov state modeling to examine nonlinear data derived from the binding isotherms and depletion curves. Some of the original studies were limited by the varying dialysis conditions, failure to account for protium-tritium exchange when using tritium-labeled tracers, and the inclusion of a narrow range of estradiol and SHBG concentrations. As described in the methods section, we took several steps to overcome these methodological concerns and to minimize their influence. Our studies provide evidence of a nonlinear, dynamic binding process involving allosteric coupling between the SHBG monomers that changes the energy landscape of both monomers and their distribution between various energy states such that the two monomers are not equivalent, even in the fully bound state and offer functional insight into the ligand-induced, intermonomeric interactions and conformational heterogeneity in SHBG.
3. RESULTS
3.1 Estradiol binding to SHBG exhibits complex interaction dynamics.
3.2 Intrinsic tryptophan emission from SHBG provides evidence that estradiol binding is multiphasic and associated with changes in the tryptophan microenvironment.
3.3 Estradiol-induced molecular rearrangement alters the conformational states of residues in the ligand-binding pockets of SHBG monomers, suggesting intermonomeric allosteric coupling.
3.4 Time-resolved lifetime fluorescence spectroscopy using bis-ANS demonstrates that estradiol binding significantly alters the global conformational state of the SHBG: E2 complex.
3.5 Dynamic cross-correlation matrix analysis shows allosteric changes in residue correlations upon estradiol binding to either of the two monomers.
3.6 Markov state models reveal dynamic allosteric conformational coupling between the SHBG monomers.
DISCUSSION
*The ligand-induced allosteric interaction between the monomers observed during estradiol's binding to SHBG may be a more general mechanism among multimeric binding proteins. We have previously found evidence of ensemble allostery in testosterone's binding to SHBG [Zakharov et al., 2015]. Our subsequent studies of testosterone's binding to human serum albumin revealed that testosterone can bind multiple (at least 6) binding sites on HSA and that testosterone binding to one site allosterically affects residues distant from the binding site (Jayaraj et al., 2020]. Variations in the apparent Kd depending on the relative concentrations of ligand and the binding protein also has been noted in studies of vitamin D binding to vitamin D binding protein. Similar dynamics in conformational ensemble and energetic repartitioning of protein populations have been shown to be functionally important in other physiologic systems, including the tetrameric hemoglobin, which exhibits intersubunit allostery in oxygen binding [Perutz, 1970; Monod et al., 1965; Koshland et al., 1966; Hilser et al., 2012; Motlagh et al., 2012].
Why would nature create such an allosteric mechanism in binding proteins?
Our findings of nonlinear dynamics of estradiol’s binding and the allosteric coupling of monomers within SHBG have potential physiological implications. First, the dynamic changes in Kd enabled by the dynamic conformational changes in SHBG upon ligand binding provide a versatile mechanism for extending the range of estradiol binding than would be possible if there were a single fixed Kd. The estradiol concentrations vary widely during different phases of the reproductive and nonreproductive phases of an individual’s life-extending from 1 to 6 pg/mL early in life and during menopause to ~ 30 to 500 pg/mL during different phases of the normal menstrual cycle to 30,000 to 40,000 pg/mL during pregnancy. Second, the nonlinear dynamics of estradiol’s binding and the allosteric coupling offer a potential mechanism for facile regulation of free hormone bioavailability under different physiologic and disease conditions. An example of this facile regulation is observed in men with hyperthyroidism some of whom develop gynecomastia. Hyperthyroidism is associated with increased levels of SHBG, and alterations in the relative ratio of free estradiol to free testosterone concentrations which have been implicated in the pathophysiology of breast enlargement in some hyperthyroid men [Chopra, 1974] Similar non-linear processes have been found in other biological systems; we speculate that this may be a more general mechanism in nature to regulate the bioavailability of nutrients and hormones. For instance, at a low partial pressure of oxygen, a relatively greater fraction of oxygen remains unbound to hemoglobin, while 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, 2021].
In conclusion, we show that estradiol binding to dimeric SHBG is a dynamic, nonlinear process that involves allosteric interaction between the two monomers of SHBG. The binding of estradiol to SHBG induces intra-molecular rearrangements in the estradiol binding pocket of the ligand-occupied monomer as well as in the binding pocket of the second monomer and alters the energy landscape of both monomers. The inter-monomeric allosteric communication is bidirectional – the binding of the second estradiol molecule also impacts the landscape and probability of conformational transitions in the first monomer, which was already bound to estradiol. Allosteric coupling in the SHBG monomers changes the energy landscape such that the two monomers are not equivalent even in the fully bound state. The allosteric interaction in the SHBG dimer may offer a potential mechanism to extend the dynamic binding range and to regulate the bioavailability of estradiol as the estradiol concentrations change several thousand-fold during the various phases of a person's life.
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