madman
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Dr. Bhasin and Dr. Jasuja most recent paper!
Estradiol binding to human serum albumin (HSA) is not a simple linear single-site interaction with a fixed dissociation constant (Kd) as assumed in traditional models (e.g., the Vermeulen/Södergård/Mazer equations for calculating free hormone fractions).
The linear law-of-mass action Vermeulen (cFTV) being the most widely used/relied on calculated method for free testosterone.
Estradiol's interaction with HSA is nonlinear and asymmetric, multi-site, dynamic and allosterically coupled.
* Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.
* The traditional view characterizes estradiol binding to HSA as linear and governed by a significantly lower association constant compared to SHBG. (28,31,33,36-38) The oft-cited model developed by Vermeulen, Södergård, and Mazer assumes a simple bimolecular equilibrium between estradiol and HSA at a single binding site, 1:1 stoichiometry, and a fixed dissociation constant (Kd). Such assumptions have formed the basis of the published and widely used equations for calculations of free hormone fractions in plasma.(39) Emerging evidence, however, suggests that the interaction between estradiol and HSA may be more complex.(32,34,35,40-47) Our previous studies of testosterone's binding to HSA revealed multiple, allosterically-coupled binding sites for testosterone on HSA that testosterone shares with free fatty acids.(48) Our preliminary studies suggested that the binding isotherm of estradiol's interaction with HSA also is not linear. Similarly, Zeginiadou et al. reported that estradiol binds to HSA in a non-linear manner and its distribution depends on the relative concentrations of these proteins. (31,49)
* In (1), the bound (Fig. 1Ai) and free estradiol (Fig. 1Aii) increased with increasing estradiol concentration, allowing determination of apparent dissociation constant (Kd) for the overall binding. The apparent Kd varied dynamically with varying estradiol concentrations (Fig.1Aiii). In (2), the bound estradiol increased with increasing HSA concentration (Fig. 1Bi), with a corresponding decrease in free estradiol (Fig. 1Bii). The apparent Kd again varied dynamically with varying HSA concentration (Fig. 1Biii). These concentration-dependent changes in apparent Kd cannot be reconciled with the legacy linear binding model in which estradiol interacts with a single, fixed-affinity site on HSA, and instead support the presence of multiple binding sites with distinct affinities and/or ligand-induced intramolecular conformational rearrangements within HSA that dynamically modulate binding-site affinity. Consistent with this interpretation, the experimentally observed free estradiol concentrations in Fig. 1Aii and Fig. 1Bii deviate substantially from values predicted by a 1:1 stoichiometric binding model with a fixed Kd, as assumed in the Vermeulen model.
* Estradiol’s binding induces conformational rearrangements in HSA that contribute to the variable apparent Kds in the protein-ligand interaction and influence the strength and kinetics of the estradiol:HSA complex formation. The presence of multiple binding sites with graded affinities - a high-affinity site in Sudlow’s Site I and two additional moderate-affinity binding sites in a contiguous cluster - explains the concentration-dependent, non-saturating binding isotherms and the dynamically varying apparent Kd. Thus, estradiol's binding to HSA is not a simple 1:1 interaction but a coordinated, multi-site dynamic process facilitated by HSA's intrinsically connected structural architecture.
Figure 6. Docking-derived estradiol binding pockets and structural-block organization of human serum albumin (HSA). (A–C) Top-ranked docking poses of estradiol bound to HSA. The ligand is shown in black stick representation. Predicted binding energies for the three highest-scoring pockets are: (A) −9.3 kcal/mol, (B) −8.8 kcal/mol, and (C) −8.7 kcal/mol. Pocket-lining residues are labelled to highlight key hydrophobic, aromatic, and charged interactions that contribute to ligand stabilization. (D) Structural-block (SB) interaction network derived from the residue–residue contact map of HSA. Nodes represent SB1–SB12, corresponding to spatially coherent residue clusters identified through network partitioning, and edges indicate significant inter-block contacts. The network highlights highly connected hubs that contribute to the protein’s internal structural communication. (E) Three-dimensional representation of HSA with structural blocks color-coded as in panel (D), showing their spatial distribution and continuity across the protein. This mapping provides the structural context for the ligand-binding pockets visualized in panels (A–C).
Abstract
Background
Circulating estradiol is predominantly protein-bound, with human serum albumin (HSA) serving as its major carrier. While traditionally considered a carrier with low affinity and readily reversible binding at a single site, the molecular details and kinetics of estradiol-HSA interactions remain incompletely understood.
Methods
We employed equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy to characterize estradiol-HSA interactions. Surface plasmon resonance (SPR) was used to elucidate the kinetics of estradiol's association and dissociation with HSA. Structural and energetic features of binding were investigated using molecular docking and structure network analyses.
Results
Binding isotherms generated using equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy revealed non-linear asymmetric binding with apparent Kd that varied as a function of estradiol and HSA concentrations, inconsistent with canonical model of low-affinity, single-site interaction characterized by a fixed Kd. Kinetic analyses by SPR revealed initial rapid association dynamics followed by a slower second phase. Molecular modeling identified a high-affinity estradiol-binding pocket in Sudlow’s Site I and two additional low-affinity sites within a highly interconnected hub of structural blocks. Spatially coordinated conformational rearrangements accompanying estradiol partitioning into the high-affinity pocket of Sudlow’s Site I and two additional moderate-affinity sites suggest an allosterically coupled binding architecture that enables albumin to actively regulate estradiol bioavailability across a broad, physiologically relevant concentration range.
Conclusion
Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.
Introduction
17β-estradiol is the principal estrogen and a key regulator of diverse physiological processes in women and men.(1-9) Under normal physiological conditions, the majority of estradiol circulates bound to carrier proteins such as sex hormone-binding globulin (SHBG) and human serum albumin (HSA), with only about 2–4% remaining in its free, unbound form.(10-15) The bioavailability of estradiol is governed by a dynamic equilibrium between its free and protein-bound forms, which is modulated by the relative concentrations and binding affinities of SHBG and HSA.(9,16-25) SHBG, owing to its high specificity and affinity for steroid hormones, is believed to serve as the principal regulator of this equilibrium.(12,26-29) HSA is reported to have substantially lower binding affinity for estradiol than SHBG and, in spite of its high plasma concentrations, its role in regulating estradiol's bioavailability and the dynamics of its binding to estradiol are incompletely understood.(12,30-35)
The traditional view characterizes estradiol binding to HSA as linear and governed by a significantly lower association constant compared to SHBG. (28,31,33,36-38) The oft-cited model developed by Vermeulen, Södergård, and Mazer assumes a simple bimolecular equilibrium between estradiol and HSA at a single binding site, 1:1 stoichiometry, and a fixed dissociation constant (Kd). Such assumptions have formed the basis of the published and widely used equations for calculations of free hormone fractions in plasma.(39) Emerging evidence, however, suggests that the interaction between estradiol and HSA may be more complex.(32,34,35,40-47) Our previous studies of testosterone's binding to HSA revealed multiple, allosterically-coupled binding sites for testosterone on HSA that testosterone shares with free fatty acids.(48) Our preliminary studies suggested that the binding isotherm of estradiol's interaction with HSA also is not linear. Similarly, Zeginiadou et al. reported that estradiol binds to HSA in a non-linear manner and its distribution depends on the relative concentrations of these proteins. (31,49)
We used equilibrium dialysis and additional biophysical methods including fluorescence quenching, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) displacement assay, and time resolved fluorescence spectroscopy to characterize the binding affinity and stoichiometry of estradiol’s binding with HSA. Experimental conditions, including temperature, pH, and buffer composition, were rigorously controlled and maintained constant throughout all assays to minimize confounding effects on protein-ligand interactions and ensure reproducibility.(42,45-47,50-53) The application of multiple orthogonal techniques enabled us to evaluate the conformity of the observed binding data with the legacy model of estradiol's binding with HSA proposed by Vermeulen, Sodergard, and Mazer which assumes a single binding site, 1:1 stoichiometry and a fixed dissociation constant (Kd).(39)
In addition, we characterized the kinetics of estradiol's association and dissociation with HSA using surface plasmon resonance (SPR). The prevailing concept of “bioavailable” estradiol, typically defined as the fraction of circulating estradiol not bound to SHBG, is based on the assumption that HSA binds estradiol with low affinity, allowing for rapid dissociation in tissue microenvironments. (28,30) Notably, the kinetic parameters governing estradiol-HSA interactions have not been measured using high-resolution temporal techniques. Our SPR experiments address this gap by providing the first direct kinetic analysis of estradiol binding to HSA.
Finally, we used molecular docking simulations and structure network analysis to further characterize the dynamics of estradiol's binding to HSA and provide important structural energetic, and mechanistic insights into estradiol-binding sites on HSA. Docking simulations identified multiple binding sites, with the top-ranked pose indicating a strong interaction. The structure network analysis highlighted HSA’s adaptability in accommodating estradiol and potential conformational changes influencing the binding affinity. Collectively, the data reported in this manuscript provide important insights into the dynamics and kinetics of estradiol's binding to HSA and conformational coupling across binding pocket residues in the HSA:estradiol complex.
Results
Binding isotherms generated using equilibrium dialysis do not conform to a single-site linear binding model with a fixed Kd
We used equilibrium dialysis to characterize the binding of estradiol to HSA, measuring the bound and free estradiol concentrations in two formats: 1) keeping the HSA concentration fixed at 500 µM (near physiological) and varying the estradiol concentration; or 2) keeping estradiol concentration fixed at 50 nM and varying the HSA concentration.
In (1), the bound (Fig. 1Ai) and free estradiol (Fig. 1Aii) increased with increasing estradiol concentration, allowing determination of apparent dissociation constant (Kd) for the overall binding. The apparent Kd varied dynamically with varying estradiol concentrations (Fig.1Aiii). In (2), the bound estradiol increased with increasing HSA concentration (Fig. 1Bi), with a corresponding decrease in free estradiol (Fig. 1Bii). The apparent Kd again varied dynamically with varying HSA concentration (Fig. 1Biii). These concentration-dependent changes in apparent Kd cannot be reconciled with the legacy linear binding model in which estradiol interacts with a single, fixed-affinity site on HSA, and instead support the presence of multiple binding sites with distinct affinities and/or ligand-induced intramolecular conformational rearrangements within HSA that dynamically modulate binding-site affinity. Consistent with this interpretation, the experimentally observed free estradiol concentrations in Fig. 1Aii and Fig. 1Bii deviate substantially from values predicted by a 1:1 stoichiometric binding model with a fixed Kd, as assumed in the Vermeulen model.
Conclusion
The integrated computational and experimental approach demonstrates that estradiol binding to HSA occurs through a coordinated, concentration-dependent engagement of multiple structurally-linked binding pockets organized around a central hub. This model successfully explains the full spectrum of non-linear binding behaviour. Estradiol’s binding induces conformational rearrangements in HSA that contribute to the variable apparent Kds in the protein-ligand interaction and influence the strength and kinetics of the estradiol:HSA complex formation. The presence of multiple binding sites with graded affinities - a high-affinity site in Sudlow’s Site I and two additional moderate-affinity binding sites in a contiguous cluster - explains the concentration-dependent, non-saturating binding isotherms and the dynamically varying apparent Kd. Thus, estradiol's binding to HSA is not a simple 1:1 interaction but a coordinated, multi-site dynamic process facilitated by HSA's intrinsically connected structural architecture.
Estradiol binding to human serum albumin (HSA) is not a simple linear single-site interaction with a fixed dissociation constant (Kd) as assumed in traditional models (e.g., the Vermeulen/Södergård/Mazer equations for calculating free hormone fractions).
The linear law-of-mass action Vermeulen (cFTV) being the most widely used/relied on calculated method for free testosterone.
Estradiol's interaction with HSA is nonlinear and asymmetric, multi-site, dynamic and allosterically coupled.
* Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.
* The traditional view characterizes estradiol binding to HSA as linear and governed by a significantly lower association constant compared to SHBG. (28,31,33,36-38) The oft-cited model developed by Vermeulen, Södergård, and Mazer assumes a simple bimolecular equilibrium between estradiol and HSA at a single binding site, 1:1 stoichiometry, and a fixed dissociation constant (Kd). Such assumptions have formed the basis of the published and widely used equations for calculations of free hormone fractions in plasma.(39) Emerging evidence, however, suggests that the interaction between estradiol and HSA may be more complex.(32,34,35,40-47) Our previous studies of testosterone's binding to HSA revealed multiple, allosterically-coupled binding sites for testosterone on HSA that testosterone shares with free fatty acids.(48) Our preliminary studies suggested that the binding isotherm of estradiol's interaction with HSA also is not linear. Similarly, Zeginiadou et al. reported that estradiol binds to HSA in a non-linear manner and its distribution depends on the relative concentrations of these proteins. (31,49)
* In (1), the bound (Fig. 1Ai) and free estradiol (Fig. 1Aii) increased with increasing estradiol concentration, allowing determination of apparent dissociation constant (Kd) for the overall binding. The apparent Kd varied dynamically with varying estradiol concentrations (Fig.1Aiii). In (2), the bound estradiol increased with increasing HSA concentration (Fig. 1Bi), with a corresponding decrease in free estradiol (Fig. 1Bii). The apparent Kd again varied dynamically with varying HSA concentration (Fig. 1Biii). These concentration-dependent changes in apparent Kd cannot be reconciled with the legacy linear binding model in which estradiol interacts with a single, fixed-affinity site on HSA, and instead support the presence of multiple binding sites with distinct affinities and/or ligand-induced intramolecular conformational rearrangements within HSA that dynamically modulate binding-site affinity. Consistent with this interpretation, the experimentally observed free estradiol concentrations in Fig. 1Aii and Fig. 1Bii deviate substantially from values predicted by a 1:1 stoichiometric binding model with a fixed Kd, as assumed in the Vermeulen model.
* Estradiol’s binding induces conformational rearrangements in HSA that contribute to the variable apparent Kds in the protein-ligand interaction and influence the strength and kinetics of the estradiol:HSA complex formation. The presence of multiple binding sites with graded affinities - a high-affinity site in Sudlow’s Site I and two additional moderate-affinity binding sites in a contiguous cluster - explains the concentration-dependent, non-saturating binding isotherms and the dynamically varying apparent Kd. Thus, estradiol's binding to HSA is not a simple 1:1 interaction but a coordinated, multi-site dynamic process facilitated by HSA's intrinsically connected structural architecture.
Figure 6. Docking-derived estradiol binding pockets and structural-block organization of human serum albumin (HSA). (A–C) Top-ranked docking poses of estradiol bound to HSA. The ligand is shown in black stick representation. Predicted binding energies for the three highest-scoring pockets are: (A) −9.3 kcal/mol, (B) −8.8 kcal/mol, and (C) −8.7 kcal/mol. Pocket-lining residues are labelled to highlight key hydrophobic, aromatic, and charged interactions that contribute to ligand stabilization. (D) Structural-block (SB) interaction network derived from the residue–residue contact map of HSA. Nodes represent SB1–SB12, corresponding to spatially coherent residue clusters identified through network partitioning, and edges indicate significant inter-block contacts. The network highlights highly connected hubs that contribute to the protein’s internal structural communication. (E) Three-dimensional representation of HSA with structural blocks color-coded as in panel (D), showing their spatial distribution and continuity across the protein. This mapping provides the structural context for the ligand-binding pockets visualized in panels (A–C).
Abstract
Background
Circulating estradiol is predominantly protein-bound, with human serum albumin (HSA) serving as its major carrier. While traditionally considered a carrier with low affinity and readily reversible binding at a single site, the molecular details and kinetics of estradiol-HSA interactions remain incompletely understood.
Methods
We employed equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy to characterize estradiol-HSA interactions. Surface plasmon resonance (SPR) was used to elucidate the kinetics of estradiol's association and dissociation with HSA. Structural and energetic features of binding were investigated using molecular docking and structure network analyses.
Results
Binding isotherms generated using equilibrium dialysis, steady-state and time-resolved fluorescence spectroscopy revealed non-linear asymmetric binding with apparent Kd that varied as a function of estradiol and HSA concentrations, inconsistent with canonical model of low-affinity, single-site interaction characterized by a fixed Kd. Kinetic analyses by SPR revealed initial rapid association dynamics followed by a slower second phase. Molecular modeling identified a high-affinity estradiol-binding pocket in Sudlow’s Site I and two additional low-affinity sites within a highly interconnected hub of structural blocks. Spatially coordinated conformational rearrangements accompanying estradiol partitioning into the high-affinity pocket of Sudlow’s Site I and two additional moderate-affinity sites suggest an allosterically coupled binding architecture that enables albumin to actively regulate estradiol bioavailability across a broad, physiologically relevant concentration range.
Conclusion
Estradiol's binding to HSA is a dynamic, multi-equilibrium process driven by ligand-induced conformational rearrangements within HSA; the binding data are inconsistent with canonical model of estradiol-HSA interaction with 1:1 stoichiometry and a fixed Kd.
Introduction
17β-estradiol is the principal estrogen and a key regulator of diverse physiological processes in women and men.(1-9) Under normal physiological conditions, the majority of estradiol circulates bound to carrier proteins such as sex hormone-binding globulin (SHBG) and human serum albumin (HSA), with only about 2–4% remaining in its free, unbound form.(10-15) The bioavailability of estradiol is governed by a dynamic equilibrium between its free and protein-bound forms, which is modulated by the relative concentrations and binding affinities of SHBG and HSA.(9,16-25) SHBG, owing to its high specificity and affinity for steroid hormones, is believed to serve as the principal regulator of this equilibrium.(12,26-29) HSA is reported to have substantially lower binding affinity for estradiol than SHBG and, in spite of its high plasma concentrations, its role in regulating estradiol's bioavailability and the dynamics of its binding to estradiol are incompletely understood.(12,30-35)
The traditional view characterizes estradiol binding to HSA as linear and governed by a significantly lower association constant compared to SHBG. (28,31,33,36-38) The oft-cited model developed by Vermeulen, Södergård, and Mazer assumes a simple bimolecular equilibrium between estradiol and HSA at a single binding site, 1:1 stoichiometry, and a fixed dissociation constant (Kd). Such assumptions have formed the basis of the published and widely used equations for calculations of free hormone fractions in plasma.(39) Emerging evidence, however, suggests that the interaction between estradiol and HSA may be more complex.(32,34,35,40-47) Our previous studies of testosterone's binding to HSA revealed multiple, allosterically-coupled binding sites for testosterone on HSA that testosterone shares with free fatty acids.(48) Our preliminary studies suggested that the binding isotherm of estradiol's interaction with HSA also is not linear. Similarly, Zeginiadou et al. reported that estradiol binds to HSA in a non-linear manner and its distribution depends on the relative concentrations of these proteins. (31,49)
We used equilibrium dialysis and additional biophysical methods including fluorescence quenching, 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid (bis-ANS) displacement assay, and time resolved fluorescence spectroscopy to characterize the binding affinity and stoichiometry of estradiol’s binding with HSA. Experimental conditions, including temperature, pH, and buffer composition, were rigorously controlled and maintained constant throughout all assays to minimize confounding effects on protein-ligand interactions and ensure reproducibility.(42,45-47,50-53) The application of multiple orthogonal techniques enabled us to evaluate the conformity of the observed binding data with the legacy model of estradiol's binding with HSA proposed by Vermeulen, Sodergard, and Mazer which assumes a single binding site, 1:1 stoichiometry and a fixed dissociation constant (Kd).(39)
In addition, we characterized the kinetics of estradiol's association and dissociation with HSA using surface plasmon resonance (SPR). The prevailing concept of “bioavailable” estradiol, typically defined as the fraction of circulating estradiol not bound to SHBG, is based on the assumption that HSA binds estradiol with low affinity, allowing for rapid dissociation in tissue microenvironments. (28,30) Notably, the kinetic parameters governing estradiol-HSA interactions have not been measured using high-resolution temporal techniques. Our SPR experiments address this gap by providing the first direct kinetic analysis of estradiol binding to HSA.
Finally, we used molecular docking simulations and structure network analysis to further characterize the dynamics of estradiol's binding to HSA and provide important structural energetic, and mechanistic insights into estradiol-binding sites on HSA. Docking simulations identified multiple binding sites, with the top-ranked pose indicating a strong interaction. The structure network analysis highlighted HSA’s adaptability in accommodating estradiol and potential conformational changes influencing the binding affinity. Collectively, the data reported in this manuscript provide important insights into the dynamics and kinetics of estradiol's binding to HSA and conformational coupling across binding pocket residues in the HSA:estradiol complex.
Results
Binding isotherms generated using equilibrium dialysis do not conform to a single-site linear binding model with a fixed Kd
We used equilibrium dialysis to characterize the binding of estradiol to HSA, measuring the bound and free estradiol concentrations in two formats: 1) keeping the HSA concentration fixed at 500 µM (near physiological) and varying the estradiol concentration; or 2) keeping estradiol concentration fixed at 50 nM and varying the HSA concentration.
In (1), the bound (Fig. 1Ai) and free estradiol (Fig. 1Aii) increased with increasing estradiol concentration, allowing determination of apparent dissociation constant (Kd) for the overall binding. The apparent Kd varied dynamically with varying estradiol concentrations (Fig.1Aiii). In (2), the bound estradiol increased with increasing HSA concentration (Fig. 1Bi), with a corresponding decrease in free estradiol (Fig. 1Bii). The apparent Kd again varied dynamically with varying HSA concentration (Fig. 1Biii). These concentration-dependent changes in apparent Kd cannot be reconciled with the legacy linear binding model in which estradiol interacts with a single, fixed-affinity site on HSA, and instead support the presence of multiple binding sites with distinct affinities and/or ligand-induced intramolecular conformational rearrangements within HSA that dynamically modulate binding-site affinity. Consistent with this interpretation, the experimentally observed free estradiol concentrations in Fig. 1Aii and Fig. 1Bii deviate substantially from values predicted by a 1:1 stoichiometric binding model with a fixed Kd, as assumed in the Vermeulen model.
Conclusion
The integrated computational and experimental approach demonstrates that estradiol binding to HSA occurs through a coordinated, concentration-dependent engagement of multiple structurally-linked binding pockets organized around a central hub. This model successfully explains the full spectrum of non-linear binding behaviour. Estradiol’s binding induces conformational rearrangements in HSA that contribute to the variable apparent Kds in the protein-ligand interaction and influence the strength and kinetics of the estradiol:HSA complex formation. The presence of multiple binding sites with graded affinities - a high-affinity site in Sudlow’s Site I and two additional moderate-affinity binding sites in a contiguous cluster - explains the concentration-dependent, non-saturating binding isotherms and the dynamically varying apparent Kd. Thus, estradiol's binding to HSA is not a simple 1:1 interaction but a coordinated, multi-site dynamic process facilitated by HSA's intrinsically connected structural architecture.
