madman
Super Moderator
Most recent paper, Bhasin and Jasuja!
* Understanding the structural arrangement of LG domain, its interaction with rest part of the protein as well as internal dynamics provides valuable insights into the molecular mechanisms governing its function as a carrier protein for sex hormones selectivity and binding preferences within the single steroid-binding site of the SHBG monomer. Collectively, this integrative approach provides a mechanistic framework to investigate how distant mutations reshape the conformational landscape of SHBG and modulate its hormone-binding behavior.
* The loop-dominant SHBG structure is critical for interactions with specific ligands. The different orientations of androgens and estrogens within the SHBG steroid-binding site hold functional significance12. This alteration in orientation influences the molecular interactions and accessibility within the binding site.
Abstract:
Sex hormone-binding globulin (SHBG), a glycoprotein in circulation, binds testosterone,m dihydrotestosterone, and estradiol with high specificity, regulating their transport and bioavailability. This function relies on long-range conformational interactions between its N terminal (NTD) and C-terminal (CTD) domains. Variations in SHBG levels or binding affinities alter free hormone concentrations, influencing reproductive and metabolic health. Despite its significance, the full-length SHBG structure and the conformational dynamics influencing hormone binding remain unclear. Deploying in-silico structural analysis, Raman spectroscopy, and network modeling, we investigated the intramolecular structural dynamics of the full length SHBG to understand how allosteric perturbations caused by natural mutations affect hormone binding and inter-residue interactions. Raman spectroscopy and in-silico analyses show that majority of the residues in SHBG (308 residues) constitute loop regions, whereas only 21% constitute beta sheet. Mutations in SHBG that alter its binding affinity, though distant from the ligand-binding pocket (LBP), induce long-range conformational changes. These mutations are clustered in flexible regions but maintain structural order through dense local interactions. Our in-silico analyses identified key substructures regulating allosteric interactions between mutationsites and ligand-binding residues. This study provides a template for further structural analyses of clinically reported mutations and their effect on hormone binding and action
1. Introduction:
Sex hormone binding globulin (SHBG) is a 45KDa glycoprotein produced primarily in the liver1,2. It associates with sex hormones- testosterone, dihydrotestosterone (DHT), and estradiol- with high affinity in the bloodstream3 and plays an important role in regulating their distribution, transport, and tissue bioavailability4. These hormones share a common binding pocket on SHBG. SHBG comprises a homodimeric assembly5, made up of two identical monomers, each consisting of 401 amino acids. SHBG’s distinct structural arrangement allows itto interact selectively with sex hormones6. Understanding the intricate interplay between SHBG and sex hormones is essential in comprehending the regulatory mechanisms governing hormonal actions in the body. Alterations in SHBG levels or its affinity for sex hormones are associated with alterations in the circulating concentrations of the unbound or the free hormone and can impact an individual’s reproductive and metabolic health7. Together, SHBG’s structure and function play an important role in modulating the bioavailability and actions of sex hormones, orchestrating a delicate hormonal balance. Monomeric structure of SHBG involves a tandem repeat of laminin G-like (LG) domains8,9, which play a crucial role in its functionality and interactions with sex hormones. The LG domain is mainly responsible for its specific binding to sex hormones, particularly testosterone, dihydrotestosterone (DHT), and estradiol10,11. This domain within SHBG create a binding groove that accommodates and interacts with the hydrophobic regions of the sex hormones. Additionally, these LG domains contribute to SHBG's stability and structural integrity11. Mutations or alterations within these LG domains can affect SHBG's binding affinity for sex hormones, potentially leading to changes in free hormone levelsand activity9. Moreover, the tandem arrangement of LG domains in SHBG allows for a range of interactions with multiple hormones within a steroid class2
Previous studies have revealed how SHBG distinguishes between androgens (such as testosterone and dihydrotestosterone) and estrogens (like estradiol), preferentially binding them in opposite orientations within the single steroid-binding site present in each SHBG monomer12.Androgens and estrogens possess distinct molecular structures and hydrophobic regions13.SHBG's steroid-binding site within the LG domains, is adaptable, enabling it to selectively recognize and bind androgens and estrogens through specific interactions within its steroid-binding pocket2,14. Several mutations in the gene encoding SHBG have been reported to impact its binding affinity for its ligands, testosterone, dihydrotestosterone (DHT), and estradiol9. These mutations in the SHBG gene could influence its binding affinity for these ligands by altering its structure, stability, or functional domains. The extant studies on dynamics of hormone bioavailability built upon findings from LBD crystal structure8,10 of truncated ligand binding domain may not accurately capture the solution dynamics and conformation flexibility across the full length SHBG. Despite previous research into SHBG’s function and ligand interactions, the full-length structural organization and conformational dynamics that govern its ligand-binding properties remains unavailable to date. A particularly unresolved and clinically significant question is how naturally occurring mutations, often located far from the ligand-binding pocket (LBP), alter SHBG's hormone-binding affinity. The spatial separation of these mutations from the LBP suggests an allosteric mechanism, but the structural and dynamic pathways underlying such long-range communication are poorly characterized. Understanding these allosteric effects is essential to elucidate how SHBG regulates hormonal bioavailability and to interpret the functional consequences of disease-associated SHBG variants.
The present work provides a comprehensive insight on how the mutations which are distant from ligand-binding domain (LBD) could alter ligand specificity. These mutations, although spatially separated from the binding pocket, are hypothesized to induce conformational changes that propagate through the protein’s structure, ultimately modulating its hormone-binding affinity. To elucidate this mechanism, we characterized the structural arrangement of SHBG monomer by which mutations impact its ligand binding. To generate a full-length model of SHBG, we performed homology modeling and validated the structure through AlphaFold comparison and Raman spectroscopy. To characterize regions of intrinsic disorder and flexibility, and to assess how mutation sites align with these dynamic regions, we conducted a comprehensive disorder propensity analysis using consensus-based predictive tools. We were particularly interested in understanding whether allosteric effects play a role in modulating the effects of mutations that are distant from the ligand binding pocket on SHBG's interactions with its ligands. In order to determine how mutation sites influence ligand-binding residues, we carried out structure network analysis, identifying key structural blocks (SBs) that mediate allosteric communication. Changes in distant regions of the protein due to mutations could potentially propagate conformationa lchanges that indirectly affect the ligand-binding site, altering its affinity for sex hormones.Therefore, to evaluate the internal flexibility and dynamic coupling of different regions within SHBG, we employed Monte Carlo simulations. Understanding the structural arrangement of LG domain, its interaction with rest part of the protein as well as internal dynamics provides valuable insights into the molecular mechanisms governing its function as a carrier protein for sex hormones selectivity and binding preferences within the single steroid-binding site of the SHBG monomer. Collectively, this integrative approach provides a mechanistic framework toinvestigate how distant mutations reshape the conformational landscape of SHBG and modulateits hormone-binding behavior.
2. Results:
As shown in Fig. 1, to accomplish our objectives, we deployed a combination of in-silico methods and spectroscopic techniques to characterize key regions in the SHBG protein structure which regulate the internal dynamics as well as ligand binding. We performed homology modelling and generated the model structure of the SHBG monomer. We verified the accuracy of our model by comparing it with the monomeric structure predicted by AlphaFold. Also, to complement the in-silico model and capture dynamic structural features, we performed Raman Spectroscopy, which provided experimental insights into the secondary structural organization and compared it with the in-silico model structure. This comparison helped us to bridge the resolution limitations of purely computational models while capturing dynamic features inaccessible to static structures. Subsequently, we analysed the structures of the DHT and estradiol-bound SHBG to investigate the residues involved in hormone binding and their spatial configuration within the protein structure. To determine the internal arrangement and mutual interactions between residues, we performed a comprehensive structure network analysis by performing normal mode analysis (NMA). The NMA included generating both all-residue structure network and community cluster network and a correlation network between amino acid residues. This approach allowed us to understand potential allosteric pathways linking distant regions to the ligand-binding site. We further studied the flexibility of SHBG monomer using MC simulation to identify key residues that regulate the structural dynamics of the protein. This integrative approach has provided important insights into critical aspects governing protein behaviour and its binding interactions with ligands. Fig. 1 represents the workflow outlined inthe manuscript.
Together, this integrative framework addresses the limitations of earlier truncated or static SHBG models by offering a full-length, dynamic perspective essential for uncovering allosteric regulation and mutation-driven effects on ligand binding.
Fig.1. Ensemble Structure Analysis Workflow to probe into SHBG structural dynamics.(A) Homology modelling was performed using the human SHBG sequence to generate a model structure of the SHBG monomer. (B) To validate our model structure, we compared it with the monomeric structure obtained from AlphaFold and further corroborated it by elucidation of secondary structural organization. using Raman Spectroscopy. (C) We analyzed ligand-bound SHBG structures to identify the protein residues interacting with DHT and estradiol and their specific interaction topology. (D) Structure network analysis was performed to reveal the distribution and interactions of residues within the 3D protein structure. (E) We used Monte Carlo simulation to investigate backbone flexibility of the SHBG monomer, revealing coupled motions of different regions/residues. (F) We identified important residues that regulate intramolecular structural dynamics in SHBG and influence ligand affinity. By integrating experimental and in-silico data, this cumulative workflow has offered a mechanistic understanding of SHBG's structural dynamics and regions important in hormone binding.
3. Discussion and Conclusion:
Our integrated approach reveals that loop-rich structure of SHBG facilitate extensive allosteric communication between mutation sites and the ligand-binding pockets. By generating the in-silico full-length SHBG structure and validating it through Raman spectroscopy, we investigated previously unresolved structural regions. Our findings showed that around 77% of the SHBG structure comprises loop regions which, despite their disordered tendencies, exhibit structural organization essential for ligand binding, inter-domain communication, and dimerization. Structure network and contact map analyses identified strong interconnections between these loop regions and the β-barrel binding site, suggesting potential allosteric regulation pathways. Notably, mutations were predominantly located within these flexible loops and, although spatially distant from the binding sites, were found to influence hormone affinity supporting the presence of long-range allosteric effects. Together, these findings highlight a structurally coordinated mechanism where loop-mediated allosteric communication modulates SHBG’sligand-binding property.
The complex interactions between proteins and their ligands influence the clinical manifestation of diverse physiological processes22. While hormone binding proteins (HBPs) and regular proteins participate in this intriguing interplay, their mechanisms and biological contexts vary distinctly. Binding pockets in HBPs have evolved to accommodate their specific hormone partners23,24. This high affinity ensures that the signal reaches the intended recipient, orchestrating specific cellular responses. HBPs, such as Sex Hormone-Binding Globulin (SHBG)14 and Corticosteroid-Binding Globulin (CBG)25,26, thyroxine-binding globulin (TBG)27,28 have evolved to bind and transport specific hormones. HBPs are classified into two main types: steroid-binding and non-steroid-binding proteins. Non-steroid-binding proteins,including various enzymes, transport proteins, and receptors, interact with a diverse range of ligands such as neurotransmitters, enzymatic substrates, and signalling molecules, playing critical roles in biological processes29-31. In contrast, the steroid-binding proteins, like SHBG and CBG, exhibit high specificity for steroid molecules such as testosterone, estrogen, and cortisol, facilitating their transport and modulating their bioavailability 32-34. This specificity enables them to effectively regulate the concentration and distribution of steroid hormones in the bloodstream, influencing physiological processes like reproduction, metabolism, and stress responses.
SHBG’s binding affinity towards specific hormones influences their distribution and accessibility, ensuring a delicate hormonal equilibrium crucial for various physiological functions. These associations highlight SHBG as a potential biomarker for diagnosing and monitoring hormonal disorders and related pathologies35-37. The loop-dominant SHBG structure is critical for interactions with specific ligands. The different orientations of androgens and estrogens within the SHBG steroid-binding site hold functional significance12. This alteration in orientation influences the molecular interactions and accessibility within the binding site.
Mutations at different sites of SHBG disrupt its hormone-binding affinity, leading to an array of health conditions9,38. Therefore, understanding the structural orchestration of SHBG, importance of its different regions from the perspective of its ligand binding as well as function is critical. But due to the unavailability of full length SHBG monomeric structure, clear understanding of the conformational dynamics associated with ligand binding and their disruptions under different mutation conditions have remained elusive and not fully understood. Unlike truncated structures, the full-length SHBG can reveal how the LBD communicates with other regions of the protein, allowing for a more accurate representation of its conformational dynamics and flexibility in solution. Additionally, full-length SHBG is vital for investigating allosteric mechanisms and how distant mutations can influence hormone bioavailability and regulatory functions. In this study,we generated in-silico SHBG model and investigated its structural arrangement and possible allosteric interactions.
Loop regions in proteins are essential components that contribute significantly to their structural dynamics, function, and versatility. SHBG, despite being comprised of 77% unstructured loop residues, shows a propensity for forming ordered substructures as observed from the disorder calculation (Fig. 4). Our Raman Spectroscopic analysis also showed that SHBG monomer contains only 13% disordered regions which validates the in-silico predictions. In monomeric SHBG, the flexible loop residues connect the N-terminal and C-terminal LG domains, facilitating dimerization2,39, inter domain interactions, and ligand binding (Fig. 2). Contact map and network analyses revealed a strong correlation between loop residues of the two domains, highlighting their role in structural communication (Fig. 1B and 7C). Also, most of the reported mutations which influence the ligand binding affinity of the protein are positioned in the flexible loop regions (Fig. 4). Notably, over 50% of reported mutations affecting ligand binding are in these flexible loops, suggesting potential allosteric crosstalk with the ligand-binding pocket inthe β-barrel region, despite the mutation sites being distant from it.
The structure network topology of the SHBG monomer revealed the interplay among residues based on pairwise interactions and interaction strength, highlighting a cross-correlation between mutation sites and ligand-binding sites. This suggests that the effects of mutations are transmitted to the hormone binding site through allosteric crosstalk. Notably, naturally occurring mutations impacting DHT and EST binding clustered in specific SBs (Supplementary Table 1), which showed dense interconnections, emphasizing their role in maintaining structural integrity. Key ligand-binding residues were positioned in SBs 3, 4, 5, 6, 7, and 10 (Table 3), with SB 5 housing 9 out of 24 pocket-forming residues and SB 6 only one. Mutations reducing DHT binding were found in SBs 2, 5, and 7, while those enhancing EST binding were in SBs 3 and 4. These SBs (2, 3, 4, 5, 6, and 7) exhibited strong interconnections, with mutation sites positioned at least 7.5 Å from ligand molecules, suggesting indirect interactions through correlated motions. This allosteric crosstalk reveals the influence of distant mutations on ligand-binding dynamics
The RMSF profile revealed variations in fluctuation intensity, highlighting distinct dynamics and stability of the LG domain compared to other regions within the protein structure (Fig. 8A). Contact map derived from RMSF identified residue islands formed through strong interactions, influencing the protein's structural dynamics. Residues 1 to 259, forming an island with maximal clique residues, demonstrated significant centrality and inter-domain contact, showing their structural importance. Most of the significant sites identified through maximal clique analysis were in SBs 2, 3, 5, 6, 7, and 8 (Supplementary Table 3), which contains the LG domain, ligand binding sites, and mutation sites. These maximal clique residues strongly interconnect with ligand-binding and mutation sites, indicating their potential role in allosteric communication and the impact of mutations on ligand binding. This interconnected cluster plays a crucial role in mediating interactions crucial for the protein's functional properties.
Our study provides a comprehensive picture on the crucial roles of loop regions, mutation sites, and SBs in defining SHBG's structural dynamics and ligand recognition. Mutation sites inflexible loop regions indirectly influence ligand-binding pockets, revealing an intricate allosteric network governing protein-ligand interactions. The full-length SHBG model presented here serves as a foundational template for future structural studies, with mechanistic insights on the implications of reported mutations and their potential downstream effects.
Our study introduces an integrative framework that brings together experimental and computational strategies to unravel the structure-function dynamics of SHBG. This approach offers several distinct advantages. First, we present, for the first time, an in-silico model of the full-length monomeric SHBG protein, enabling detailed analysis of inter-domain interactions and structural communication that are absent in truncated versions. Second, Raman spectroscopic validation of secondary structure complements disorder predictions, enhancing confidence in model accuracy and structural interpretations. Third, by means of structure network and correlation analyses, we mapped previously uncharacterized allosteric pathways connectingdistant mutation clusters with the hormone-binding pocket. Finally, the integration of RMSF profiling, contact maps, and maximal clique analysis offers a comprehensive view of SHBG's conformational landscape, identifying key residue islands involved in stability and ligand responsiveness. Together, these strengths provide a foundational basis for understanding the SHBG function in health and disease, with implications for targeted drug design and biomarker discovery.
Absence of functional experimental assays probing into conformational dynamics associated with the SHBG and its mutants is a limitation in the current study. Future work would aim at studying the mutant structures that effect ligand binding and investigating how the mutations alter critical contacts in the ligand binding pocket. Further, using in-silico approaches we would study different mutant structures of SHBG to understand how the loop movements modulate the pocket formation for specific hormones. Additionally, combining experimental and computational strategies using both ligand-bound and unbound structures would help to identify intermediate protein conformations and provide a deeper understanding of SHBG's conformational landscape. Such studies would provide insights into the allosteric regulation mechanisms and might inform the development of targeted therapeutics that modulate SHBG function.
* Understanding the structural arrangement of LG domain, its interaction with rest part of the protein as well as internal dynamics provides valuable insights into the molecular mechanisms governing its function as a carrier protein for sex hormones selectivity and binding preferences within the single steroid-binding site of the SHBG monomer. Collectively, this integrative approach provides a mechanistic framework to investigate how distant mutations reshape the conformational landscape of SHBG and modulate its hormone-binding behavior.
* The loop-dominant SHBG structure is critical for interactions with specific ligands. The different orientations of androgens and estrogens within the SHBG steroid-binding site hold functional significance12. This alteration in orientation influences the molecular interactions and accessibility within the binding site.
Abstract:
Sex hormone-binding globulin (SHBG), a glycoprotein in circulation, binds testosterone,m dihydrotestosterone, and estradiol with high specificity, regulating their transport and bioavailability. This function relies on long-range conformational interactions between its N terminal (NTD) and C-terminal (CTD) domains. Variations in SHBG levels or binding affinities alter free hormone concentrations, influencing reproductive and metabolic health. Despite its significance, the full-length SHBG structure and the conformational dynamics influencing hormone binding remain unclear. Deploying in-silico structural analysis, Raman spectroscopy, and network modeling, we investigated the intramolecular structural dynamics of the full length SHBG to understand how allosteric perturbations caused by natural mutations affect hormone binding and inter-residue interactions. Raman spectroscopy and in-silico analyses show that majority of the residues in SHBG (308 residues) constitute loop regions, whereas only 21% constitute beta sheet. Mutations in SHBG that alter its binding affinity, though distant from the ligand-binding pocket (LBP), induce long-range conformational changes. These mutations are clustered in flexible regions but maintain structural order through dense local interactions. Our in-silico analyses identified key substructures regulating allosteric interactions between mutationsites and ligand-binding residues. This study provides a template for further structural analyses of clinically reported mutations and their effect on hormone binding and action
1. Introduction:
Sex hormone binding globulin (SHBG) is a 45KDa glycoprotein produced primarily in the liver1,2. It associates with sex hormones- testosterone, dihydrotestosterone (DHT), and estradiol- with high affinity in the bloodstream3 and plays an important role in regulating their distribution, transport, and tissue bioavailability4. These hormones share a common binding pocket on SHBG. SHBG comprises a homodimeric assembly5, made up of two identical monomers, each consisting of 401 amino acids. SHBG’s distinct structural arrangement allows itto interact selectively with sex hormones6. Understanding the intricate interplay between SHBG and sex hormones is essential in comprehending the regulatory mechanisms governing hormonal actions in the body. Alterations in SHBG levels or its affinity for sex hormones are associated with alterations in the circulating concentrations of the unbound or the free hormone and can impact an individual’s reproductive and metabolic health7. Together, SHBG’s structure and function play an important role in modulating the bioavailability and actions of sex hormones, orchestrating a delicate hormonal balance. Monomeric structure of SHBG involves a tandem repeat of laminin G-like (LG) domains8,9, which play a crucial role in its functionality and interactions with sex hormones. The LG domain is mainly responsible for its specific binding to sex hormones, particularly testosterone, dihydrotestosterone (DHT), and estradiol10,11. This domain within SHBG create a binding groove that accommodates and interacts with the hydrophobic regions of the sex hormones. Additionally, these LG domains contribute to SHBG's stability and structural integrity11. Mutations or alterations within these LG domains can affect SHBG's binding affinity for sex hormones, potentially leading to changes in free hormone levelsand activity9. Moreover, the tandem arrangement of LG domains in SHBG allows for a range of interactions with multiple hormones within a steroid class2
Previous studies have revealed how SHBG distinguishes between androgens (such as testosterone and dihydrotestosterone) and estrogens (like estradiol), preferentially binding them in opposite orientations within the single steroid-binding site present in each SHBG monomer12.Androgens and estrogens possess distinct molecular structures and hydrophobic regions13.SHBG's steroid-binding site within the LG domains, is adaptable, enabling it to selectively recognize and bind androgens and estrogens through specific interactions within its steroid-binding pocket2,14. Several mutations in the gene encoding SHBG have been reported to impact its binding affinity for its ligands, testosterone, dihydrotestosterone (DHT), and estradiol9. These mutations in the SHBG gene could influence its binding affinity for these ligands by altering its structure, stability, or functional domains. The extant studies on dynamics of hormone bioavailability built upon findings from LBD crystal structure8,10 of truncated ligand binding domain may not accurately capture the solution dynamics and conformation flexibility across the full length SHBG. Despite previous research into SHBG’s function and ligand interactions, the full-length structural organization and conformational dynamics that govern its ligand-binding properties remains unavailable to date. A particularly unresolved and clinically significant question is how naturally occurring mutations, often located far from the ligand-binding pocket (LBP), alter SHBG's hormone-binding affinity. The spatial separation of these mutations from the LBP suggests an allosteric mechanism, but the structural and dynamic pathways underlying such long-range communication are poorly characterized. Understanding these allosteric effects is essential to elucidate how SHBG regulates hormonal bioavailability and to interpret the functional consequences of disease-associated SHBG variants.
The present work provides a comprehensive insight on how the mutations which are distant from ligand-binding domain (LBD) could alter ligand specificity. These mutations, although spatially separated from the binding pocket, are hypothesized to induce conformational changes that propagate through the protein’s structure, ultimately modulating its hormone-binding affinity. To elucidate this mechanism, we characterized the structural arrangement of SHBG monomer by which mutations impact its ligand binding. To generate a full-length model of SHBG, we performed homology modeling and validated the structure through AlphaFold comparison and Raman spectroscopy. To characterize regions of intrinsic disorder and flexibility, and to assess how mutation sites align with these dynamic regions, we conducted a comprehensive disorder propensity analysis using consensus-based predictive tools. We were particularly interested in understanding whether allosteric effects play a role in modulating the effects of mutations that are distant from the ligand binding pocket on SHBG's interactions with its ligands. In order to determine how mutation sites influence ligand-binding residues, we carried out structure network analysis, identifying key structural blocks (SBs) that mediate allosteric communication. Changes in distant regions of the protein due to mutations could potentially propagate conformationa lchanges that indirectly affect the ligand-binding site, altering its affinity for sex hormones.Therefore, to evaluate the internal flexibility and dynamic coupling of different regions within SHBG, we employed Monte Carlo simulations. Understanding the structural arrangement of LG domain, its interaction with rest part of the protein as well as internal dynamics provides valuable insights into the molecular mechanisms governing its function as a carrier protein for sex hormones selectivity and binding preferences within the single steroid-binding site of the SHBG monomer. Collectively, this integrative approach provides a mechanistic framework toinvestigate how distant mutations reshape the conformational landscape of SHBG and modulateits hormone-binding behavior.
2. Results:
As shown in Fig. 1, to accomplish our objectives, we deployed a combination of in-silico methods and spectroscopic techniques to characterize key regions in the SHBG protein structure which regulate the internal dynamics as well as ligand binding. We performed homology modelling and generated the model structure of the SHBG monomer. We verified the accuracy of our model by comparing it with the monomeric structure predicted by AlphaFold. Also, to complement the in-silico model and capture dynamic structural features, we performed Raman Spectroscopy, which provided experimental insights into the secondary structural organization and compared it with the in-silico model structure. This comparison helped us to bridge the resolution limitations of purely computational models while capturing dynamic features inaccessible to static structures. Subsequently, we analysed the structures of the DHT and estradiol-bound SHBG to investigate the residues involved in hormone binding and their spatial configuration within the protein structure. To determine the internal arrangement and mutual interactions between residues, we performed a comprehensive structure network analysis by performing normal mode analysis (NMA). The NMA included generating both all-residue structure network and community cluster network and a correlation network between amino acid residues. This approach allowed us to understand potential allosteric pathways linking distant regions to the ligand-binding site. We further studied the flexibility of SHBG monomer using MC simulation to identify key residues that regulate the structural dynamics of the protein. This integrative approach has provided important insights into critical aspects governing protein behaviour and its binding interactions with ligands. Fig. 1 represents the workflow outlined inthe manuscript.
Together, this integrative framework addresses the limitations of earlier truncated or static SHBG models by offering a full-length, dynamic perspective essential for uncovering allosteric regulation and mutation-driven effects on ligand binding.
Fig.1. Ensemble Structure Analysis Workflow to probe into SHBG structural dynamics.(A) Homology modelling was performed using the human SHBG sequence to generate a model structure of the SHBG monomer. (B) To validate our model structure, we compared it with the monomeric structure obtained from AlphaFold and further corroborated it by elucidation of secondary structural organization. using Raman Spectroscopy. (C) We analyzed ligand-bound SHBG structures to identify the protein residues interacting with DHT and estradiol and their specific interaction topology. (D) Structure network analysis was performed to reveal the distribution and interactions of residues within the 3D protein structure. (E) We used Monte Carlo simulation to investigate backbone flexibility of the SHBG monomer, revealing coupled motions of different regions/residues. (F) We identified important residues that regulate intramolecular structural dynamics in SHBG and influence ligand affinity. By integrating experimental and in-silico data, this cumulative workflow has offered a mechanistic understanding of SHBG's structural dynamics and regions important in hormone binding.
3. Discussion and Conclusion:
Our integrated approach reveals that loop-rich structure of SHBG facilitate extensive allosteric communication between mutation sites and the ligand-binding pockets. By generating the in-silico full-length SHBG structure and validating it through Raman spectroscopy, we investigated previously unresolved structural regions. Our findings showed that around 77% of the SHBG structure comprises loop regions which, despite their disordered tendencies, exhibit structural organization essential for ligand binding, inter-domain communication, and dimerization. Structure network and contact map analyses identified strong interconnections between these loop regions and the β-barrel binding site, suggesting potential allosteric regulation pathways. Notably, mutations were predominantly located within these flexible loops and, although spatially distant from the binding sites, were found to influence hormone affinity supporting the presence of long-range allosteric effects. Together, these findings highlight a structurally coordinated mechanism where loop-mediated allosteric communication modulates SHBG’sligand-binding property.
The complex interactions between proteins and their ligands influence the clinical manifestation of diverse physiological processes22. While hormone binding proteins (HBPs) and regular proteins participate in this intriguing interplay, their mechanisms and biological contexts vary distinctly. Binding pockets in HBPs have evolved to accommodate their specific hormone partners23,24. This high affinity ensures that the signal reaches the intended recipient, orchestrating specific cellular responses. HBPs, such as Sex Hormone-Binding Globulin (SHBG)14 and Corticosteroid-Binding Globulin (CBG)25,26, thyroxine-binding globulin (TBG)27,28 have evolved to bind and transport specific hormones. HBPs are classified into two main types: steroid-binding and non-steroid-binding proteins. Non-steroid-binding proteins,including various enzymes, transport proteins, and receptors, interact with a diverse range of ligands such as neurotransmitters, enzymatic substrates, and signalling molecules, playing critical roles in biological processes29-31. In contrast, the steroid-binding proteins, like SHBG and CBG, exhibit high specificity for steroid molecules such as testosterone, estrogen, and cortisol, facilitating their transport and modulating their bioavailability 32-34. This specificity enables them to effectively regulate the concentration and distribution of steroid hormones in the bloodstream, influencing physiological processes like reproduction, metabolism, and stress responses.
SHBG’s binding affinity towards specific hormones influences their distribution and accessibility, ensuring a delicate hormonal equilibrium crucial for various physiological functions. These associations highlight SHBG as a potential biomarker for diagnosing and monitoring hormonal disorders and related pathologies35-37. The loop-dominant SHBG structure is critical for interactions with specific ligands. The different orientations of androgens and estrogens within the SHBG steroid-binding site hold functional significance12. This alteration in orientation influences the molecular interactions and accessibility within the binding site.
Mutations at different sites of SHBG disrupt its hormone-binding affinity, leading to an array of health conditions9,38. Therefore, understanding the structural orchestration of SHBG, importance of its different regions from the perspective of its ligand binding as well as function is critical. But due to the unavailability of full length SHBG monomeric structure, clear understanding of the conformational dynamics associated with ligand binding and their disruptions under different mutation conditions have remained elusive and not fully understood. Unlike truncated structures, the full-length SHBG can reveal how the LBD communicates with other regions of the protein, allowing for a more accurate representation of its conformational dynamics and flexibility in solution. Additionally, full-length SHBG is vital for investigating allosteric mechanisms and how distant mutations can influence hormone bioavailability and regulatory functions. In this study,we generated in-silico SHBG model and investigated its structural arrangement and possible allosteric interactions.
Loop regions in proteins are essential components that contribute significantly to their structural dynamics, function, and versatility. SHBG, despite being comprised of 77% unstructured loop residues, shows a propensity for forming ordered substructures as observed from the disorder calculation (Fig. 4). Our Raman Spectroscopic analysis also showed that SHBG monomer contains only 13% disordered regions which validates the in-silico predictions. In monomeric SHBG, the flexible loop residues connect the N-terminal and C-terminal LG domains, facilitating dimerization2,39, inter domain interactions, and ligand binding (Fig. 2). Contact map and network analyses revealed a strong correlation between loop residues of the two domains, highlighting their role in structural communication (Fig. 1B and 7C). Also, most of the reported mutations which influence the ligand binding affinity of the protein are positioned in the flexible loop regions (Fig. 4). Notably, over 50% of reported mutations affecting ligand binding are in these flexible loops, suggesting potential allosteric crosstalk with the ligand-binding pocket inthe β-barrel region, despite the mutation sites being distant from it.
The structure network topology of the SHBG monomer revealed the interplay among residues based on pairwise interactions and interaction strength, highlighting a cross-correlation between mutation sites and ligand-binding sites. This suggests that the effects of mutations are transmitted to the hormone binding site through allosteric crosstalk. Notably, naturally occurring mutations impacting DHT and EST binding clustered in specific SBs (Supplementary Table 1), which showed dense interconnections, emphasizing their role in maintaining structural integrity. Key ligand-binding residues were positioned in SBs 3, 4, 5, 6, 7, and 10 (Table 3), with SB 5 housing 9 out of 24 pocket-forming residues and SB 6 only one. Mutations reducing DHT binding were found in SBs 2, 5, and 7, while those enhancing EST binding were in SBs 3 and 4. These SBs (2, 3, 4, 5, 6, and 7) exhibited strong interconnections, with mutation sites positioned at least 7.5 Å from ligand molecules, suggesting indirect interactions through correlated motions. This allosteric crosstalk reveals the influence of distant mutations on ligand-binding dynamics
The RMSF profile revealed variations in fluctuation intensity, highlighting distinct dynamics and stability of the LG domain compared to other regions within the protein structure (Fig. 8A). Contact map derived from RMSF identified residue islands formed through strong interactions, influencing the protein's structural dynamics. Residues 1 to 259, forming an island with maximal clique residues, demonstrated significant centrality and inter-domain contact, showing their structural importance. Most of the significant sites identified through maximal clique analysis were in SBs 2, 3, 5, 6, 7, and 8 (Supplementary Table 3), which contains the LG domain, ligand binding sites, and mutation sites. These maximal clique residues strongly interconnect with ligand-binding and mutation sites, indicating their potential role in allosteric communication and the impact of mutations on ligand binding. This interconnected cluster plays a crucial role in mediating interactions crucial for the protein's functional properties.
Our study provides a comprehensive picture on the crucial roles of loop regions, mutation sites, and SBs in defining SHBG's structural dynamics and ligand recognition. Mutation sites inflexible loop regions indirectly influence ligand-binding pockets, revealing an intricate allosteric network governing protein-ligand interactions. The full-length SHBG model presented here serves as a foundational template for future structural studies, with mechanistic insights on the implications of reported mutations and their potential downstream effects.
Our study introduces an integrative framework that brings together experimental and computational strategies to unravel the structure-function dynamics of SHBG. This approach offers several distinct advantages. First, we present, for the first time, an in-silico model of the full-length monomeric SHBG protein, enabling detailed analysis of inter-domain interactions and structural communication that are absent in truncated versions. Second, Raman spectroscopic validation of secondary structure complements disorder predictions, enhancing confidence in model accuracy and structural interpretations. Third, by means of structure network and correlation analyses, we mapped previously uncharacterized allosteric pathways connectingdistant mutation clusters with the hormone-binding pocket. Finally, the integration of RMSF profiling, contact maps, and maximal clique analysis offers a comprehensive view of SHBG's conformational landscape, identifying key residue islands involved in stability and ligand responsiveness. Together, these strengths provide a foundational basis for understanding the SHBG function in health and disease, with implications for targeted drug design and biomarker discovery.
Absence of functional experimental assays probing into conformational dynamics associated with the SHBG and its mutants is a limitation in the current study. Future work would aim at studying the mutant structures that effect ligand binding and investigating how the mutations alter critical contacts in the ligand binding pocket. Further, using in-silico approaches we would study different mutant structures of SHBG to understand how the loop movements modulate the pocket formation for specific hormones. Additionally, combining experimental and computational strategies using both ligand-bound and unbound structures would help to identify intermediate protein conformations and provide a deeper understanding of SHBG's conformational landscape. Such studies would provide insights into the allosteric regulation mechanisms and might inform the development of targeted therapeutics that modulate SHBG function.
