Molecular interactions between sex hormone–binding globulin and nonsteroidal ligands that enhance androgen activity

Abstract

Sex hormone–binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two nonsteroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (−)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast, a synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71–1.80 Å resolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other nonsteroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ∼20-fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol-dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how nonsteroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids. Sex hormone–binding globulin (SHBG) determines the equilibrium between free and protein-bound androgens and estrogens in the blood and regulates their access to target tissues. Using crystallographic approaches and radiolabeled competitive binding-capacity assays, we report here how two nonsteroidal compounds bind to human SHBG, and how they influence androgen activity in cell culture. We found that one of these compounds, (−)3,4-divanillyltetrahydrofuran (DVT), present in stinging nettle root extracts and used as a nutraceutical, binds SHBG with relatively low affinity. By contrast, a synthetic compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI), bound SHBG with an affinity similar to that of testosterone and estradiol. Crystal structures of SHBG in complex with DVT or IPI at 1.71–1.80 Å resolutions revealed their unique orientations in the SHBG ligand-binding pocket and suggested opportunities for the design of other nonsteroidal ligands of SHBG. As observed for estradiol but not testosterone, IPI binding to SHBG was reduced by ∼20-fold in the presence of zinc, whereas DVT binding was almost completely lost. Estradiol-dependent fibulin-2 interactions with SHBG similarly occurred for IPI-bound SHBG, but not with DVT-bound SHBG. Both DVT and IPI increased the activity of testosterone in a cell culture androgen reporter system by competitively displacing testosterone from SHBG. These findings indicate how nonsteroidal ligands of SHBG maybe designed to modulate the bioavailability of sex steroids. Steroid hormone bioavailability is delineated by the “free hormone hypothesis” and its underlying tenet that steroids only enter cells when they are not bound by plasma proteins (1Mendel C.M. The free hormone hypothesis: A physiologically based mathematical model.Endocr. Rev. 1989; 10 (2673754): 232-27410.1210/edrv-10-3-232Crossref PubMed Scopus (747) Google Scholar). Sex hormone–binding globulin (SHBG) 2The abbreviations used are: SHBGsex hormone binding globulinDHT5α-dihydrotestosteroneDVT(−)3,4-divanillyltetrahydrofuranIPI3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indoleLGlaminin GRMSDroot mean square deviationRBArelative binding affinityDCCdextran-coated charcoal. is produced by hepatocytes and it binds biologically active androgens and estrogens, thereby regulating their plasma levels and bioavailability (2Hammond G.L. Plasma steroid-binding proteins: Primary gatekeepers of steroid hormone action.J. Endocrinol. 2016; 230 (27113851): R13-R2510.1530/JOE-16-0070Crossref PubMed Scopus (169) Google Scholar). Human SHBG is a homodimer, with each monomer comprising two laminin G (LG) domains (3Grishkovskaya I. Avvakumov G.V. Sklenar G. Dales D. Hammond G.L. Muller Y.A. Crystal structure of human sex hormone-binding globulin: Steroid transport by a laminin G-like domain.EMBO J. 2000; 19 (10675319): 504-51210.1093/emboj/19.4.504Crossref PubMed Scopus (131) Google Scholar). The N-terminal LG domain contains a steroid-binding site and the dimerization interface (3Grishkovskaya I. Avvakumov G.V. Sklenar G. Dales D. Hammond G.L. Muller Y.A. Crystal structure of human sex hormone-binding globulin: Steroid transport by a laminin G-like domain.EMBO J. 2000; 19 (10675319): 504-51210.1093/emboj/19.4.504Crossref PubMed Scopus (131) Google Scholar, 4Avvakumov G.V. Grishkovskaya I. Muller Y.A. Hammond G.L. Resolution of the human sex hormone-binding globulin dimer interface and evidence for two steroid-binding sites per homodimer.J. Biol. Chem. 2001; 276 (11457864): 34453-3445710.1074/jbc.M106274200Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The dimerization of SHBG depends on the occupancy of a calcium-binding site within the N-terminal LG domain and dimerization is stabilized by the binding of steroid ligands (5Bocchinfuso W.P. Hammond G.L. Steroid-binding and dimerization domains of human sex hormone-binding globulin partially overlap: Steroids and Ca2+ stabilize dimer formation.Biochemistry. 1994; 33 (8075062): 10622-1062910.1021/bi00201a008Crossref PubMed Scopus (39) Google Scholar). The carboxyl-terminal LG domain plays no role in steroid binding or dimerization but contains two N-linked glycosylation sites, the utilization of which may prolong its plasma half-life (6Cousin P. Déchaud H. Grenot C. Lejeune H. Pugeat M. Human variant sex hormone-binding globulin (SHBG) with an additional carbohydrate chain has a reduced clearance rate in rabbit.J. Clin. Endocrinol. Metab. 1998; 83 (9435448): 235-24010.1210/jcem.83.1.4515Crossref PubMed Scopus (73) Google Scholar, 7Cousin P. Déchaud H. Grenot C. Lejeune H. Hammond G.L. Pugeat M. Influence of glycosylation on the clearance of recombinant human sex hormone-binding globulin from rabbit blood.J. Steroid Biochem. Mol. Biol. 1999; 70 (10622399): 115-12110.1016/S0960-0760(99)00101-6Crossref PubMed Scopus (29) Google Scholar). sex hormone binding globulin 5α-dihydrotestosterone (−)3,4-divanillyltetrahydrofuran 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole laminin G root mean square deviation relative binding affinity dextran-coated charcoal. The active androgen, 5α-dihydrotestosterone (DHT), has the highest affinity ∼1 nm Kd for SHBG followed by testosterone and estradiol, which bind with ∼5 and ∼20 times lower affinities, respectively, when compared with DHT (8Dunn J.F. Nisula B.C. Rodbard D. Transport of steroid hormones: Binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma.J. Clin. Endocrinol. Metab. 1981; 53 (7195404): 58-6810.1210/jcem-53-1-58Crossref PubMed Scopus (1021) Google Scholar). Androgens and estrogens are positioned in opposite orientations within the same hydrophobic pocket in crystal structures of the N-terminal LG domain of SHBG such that the steroid ring A of androgens is buried within the protein near Ser-42, whereas the ring D of estrogens resides in this position, and this steroid class–dependent difference in their orientation causes minor structural variation in the protein (9Grishkovskaya I. Avvakumov G.V. Hammond G.L. Catalano M.G. Muller Y.A. Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.J. Biol. Chem. 2002; 277 (12065592): 32086-3209310.1074/jbc.M203999200Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Notably, a flexible loop region formed by residues 130–135 appears to form a lid over an entrance to the SHBG steroid-binding pocket. This loop region usually appears disordered in crystal structures when androgens are present in the binding site but is more ordered when estradiol is bound so that key residues within it interact with this steroid (9Grishkovskaya I. Avvakumov G.V. Hammond G.L. Catalano M.G. Muller Y.A. Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.J. Biol. Chem. 2002; 277 (12065592): 32086-3209310.1074/jbc.M203999200Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 10Avvakumov G.V. Grishkovskaya I. Muller Y.A. Hammond G.L. Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol.J. Biol. Chem. 2002; 277 (12228253): 45219-4522510.1074/jbc.M207762200Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Occupancy of a zinc-binding site in this region of human SHBG also alters the positioning of this loop region and the binding affinity of estrogens (10Avvakumov G.V. Grishkovskaya I. Muller Y.A. Hammond G.L. Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol.J. Biol. Chem. 2002; 277 (12228253): 45219-4522510.1074/jbc.M207762200Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 11Avvakumov G.V. Muller Y.A. Hammond G.L. Steroid-binding specificity of human sex hormone-binding globulin is influenced by occupancy of a zinc-binding site.J. Biol. Chem. 2000; 275 (10859323): 25920-2592510.1074/jbc.M004484200Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 12Grishkovskaya I. Avvakumov G.V. Hammond G.L. Muller Y.A. Resolution of a disordered region at the entrance of the human sex hormone-binding globulin steroid-binding site.J. Mol. Biol. 2002; 318 (12054810): 621-62610.1016/S0022-2836(02)00169-9Crossref PubMed Scopus (16) Google Scholar). Evidence that this flexible loop region serves as the main portal for sex steroids is largely circumstantial. However, an mAb binds an epitope in this region, and presumably constrains the mobility of the loop, and this appears to prevent the movement of DHT into and out of the steroid-binding site, whereas the binding of estradiol is only marginally affected (13Wu T.S. Hammond G.L. Naturally occurring mutants inform SHBG structure and function.Mol. Endocrinol. 2014; 28 (24892637): 1026-103810.1210/me.2014-1058Crossref PubMed Scopus (26) Google Scholar). This led to the suggestion that estradiol has an alternative route of entry into or exit from the steroid-binding site when compared with DHT (13Wu T.S. Hammond G.L. Naturally occurring mutants inform SHBG structure and function.Mol. Endocrinol. 2014; 28 (24892637): 1026-103810.1210/me.2014-1058Crossref PubMed Scopus (26) Google Scholar). If the steroid-binding pocket of SHBG has multiple entrances, it is possible that other lipophilic ligands of SHBG (14Avvakumov G.V. Cherkasov A. Muller Y.A. Hammond G.L. Structural analyses of sex hormone-binding globulin reveal novel ligands and function.Mol. Cell Endocrinol. 2010; 316 (19748550): 13-2310.1016/j.mce.2009.09.005Crossref PubMed Scopus (62) Google Scholar) access it differently and reside within it in unique orientations. Numerous nonsteroidal ligands of SHBG have been identified, including several endocrine disrupting compounds (14Avvakumov G.V. Cherkasov A. Muller Y.A. Hammond G.L. Structural analyses of sex hormone-binding globulin reveal novel ligands and function.Mol. Cell Endocrinol. 2010; 316 (19748550): 13-2310.1016/j.mce.2009.09.005Crossref PubMed Scopus (62) Google Scholar, 15Hodgert Jury H. Zacharewski T.R. Hammond G.L. Interactions between human plasma sex hormone-binding globulin and xenobiotic ligands.J. Steroid Biochem. Mol. Biol. 2000; 75 (11226833): 167-17610.1016/S0960-0760(00)00168-0Crossref PubMed Scopus (50) Google Scholar). Among these, (−)3,4-divanillyltetrahydrofuran (DVT) is a natural compound present in stinging nettle root extracts used for the treatment of benign prostatic hypertrophy (Fig. 1) (16Schöttner M. Gansser D. Spiteller G. Lignans from the roots of Urtica dioica and their metabolites bind to human sex hormone binding globulin (SHBG).Planta Med. 1997; 63 (9434605): 529-53210.1055/s-2006-957756Crossref PubMed Scopus (88) Google Scholar, 17Schöttner M. Spiteller G. Gansser D. Lignans interfering with 5α-dihydrotestosterone binding to human sex hormone-binding globulin.J. Nat. Prod. 1998; 61 (9461660): 119-12110.1021/np9701743Crossref PubMed Scopus (52) Google Scholar) and is widely used as a nutraceutical with anabolic properties (18McDonald T.J. Perry M.H. Jones A.G. Donohoe M. Salzmann M.B. O'Connor J. A novel case of a raised testosterone and LH in a young man.Clin. Chim. Acta. 2011; 412 (21756889): 1999-200110.1016/j.cca.2011.06.028Crossref PubMed Scopus (1) Google Scholar). We therefore set out to determine the molecular interactions between SHBG and DVT, as well as a novel nonsteroidal compound, 3-(1H-imidazol-1-ylmethyl)-2phenyl-1H-indole (IPI) provided by Pfizer (PF-0102478) as a potential SHBG ligand (Fig. 1), and we have studied their ability to displace androgens from SHBG and to enhance their bioavailability. The N-terminal LG domain of human SHBG E176K crystallizes within 24 h into large 3-dimensional diamond-shaped crystals in space group P4322 that provide reliable data collection. The crystal structure of estradiol-bound SHBG E176K was determined at 1.73 Å and solved by molecular replacement with the published N-terminal LG domain SHBG structure in complex with estradiol (9Grishkovskaya I. Avvakumov G.V. Hammond G.L. Catalano M.G. Muller Y.A. Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.J. Biol. Chem. 2002; 277 (12065592): 32086-3209310.1074/jbc.M203999200Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Data collection parameters and refinement statistics are presented in Table 1. When these structures are superimposed, the steroid-binding pocket, the flexible loop region that covers it, and the position and orientation of estradiol do not differ from each other (Fig. 2A). Structural alignment of α carbon atoms between estradiol-bound E176K and previously published estradiol-bound WT SHBG structures resulted in a RMSD of 1.25 Å. Residues involved in steroid binding are positioned as expected (Fig. 2B). Although the orientation of Trp-84 differs in position, its electron density in SHBG E176K is poorly defined, indicating that there may be a secondary conformation for this residue. In addition, the side chain of Lys-173 is displaced and a suspected water molecule is organized by hydrogen bonding between Lys-173 and Lys-176, when compared with Glu-176 (Fig. 2C), and this change is located near residues (Leu-171–Lys-173) that are displaced when estradiol is bound (10Avvakumov G.V. Grishkovskaya I. Muller Y.A. Hammond G.L. Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol.J. Biol. Chem. 2002; 277 (12228253): 45219-4522510.1074/jbc.M207762200Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar).Table 1Data collection and refinement statisticsE2-bound E176K SHBGDVT-bound E176K SHBGIPI-bound E176K SHBGData CollectionSpace groupP 43 2 2P 43 2 2P 43 2 2Cell dimensionsa, b, c (Å)51.87, 51.87, 149.651.95, 51.95, 148.452.24, 52.24, 147.9Total reflections44,460 (4330)39,386 (3802)46,065 (4470)Unique reflections22,230 (2165)19,693 (1901)23,048 (2235)Resolution range42.6–1.73 (1.79–1.73)29.5–1.80 (1.86–1.80)25.7–1.71 (1.77–1.71)Rmerge (%)1.3 (18.0)1.2 (21.0)1.3 (13.0)CC1/21 (0.929)1 (0.910)1 (0.959)CC*1 (0.981)1 (0.976)1 (0.989)I/σI23.0 (4.2)23.0 (3.1)24.0 (4.4)Completeness (%)99.6 (100)99.9 (100)99.7 (100)RefinementResolution42.6–1.73 (1.79–1.73)29.5–1.80 (1.86–1.80)25.7–1.71 (1.77–1.71)Rwork/Rfree (%)18.4/22.120.3/23.118.5/21.6Number of atomsProtein139513561412Ligands212622Water186110121Average B-factorsProtein28.241.232.0Ligands19.545.827.4Water34.842.837.4RMSD bond lengths (Å)0.0080.0030.006RMSD bond angles (°)1.050.750.84Ramachandran statisticsFavored regions (%)95.995.897.1Allowed regions (%)4.14.22.9Outliers (%)000Rotamer outliers (%)00.71.3Clash score1.071.472.11MolProbity score1.11 (99th percentile of similar resolution structures)1.20 (99th percentile of similar resolution structures)1.14 (99th percentile of similar resolution structures) Open table in a new tab The crystal structure of SHBG E176K in complex with DVT was determined at 1.80 Å and solved by molecular replacement with the estradiol-bound SHBG E176K structure. This shows that DVT is bound at same site as sex steroids but occupies more of the hydrophobic pocket, while maintaining key residue interactions (Fig. 3A). No major changes to the globular structure were observed when compared with other SHBG crystal structures with several steroid ligands (Fig. 3A) (3Grishkovskaya I. Avvakumov G.V. Sklenar G. Dales D. Hammond G.L. Muller Y.A. Crystal structure of human sex hormone-binding globulin: Steroid transport by a laminin G-like domain.EMBO J. 2000; 19 (10675319): 504-51210.1093/emboj/19.4.504Crossref PubMed Scopus (131) Google Scholar, 9Grishkovskaya I. Avvakumov G.V. Hammond G.L. Catalano M.G. Muller Y.A. Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.J. Biol. Chem. 2002; 277 (12065592): 32086-3209310.1074/jbc.M203999200Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 10Avvakumov G.V. Grishkovskaya I. Muller Y.A. Hammond G.L. Crystal structure of human sex hormone-binding globulin in complex with 2-methoxyestradiol reveals the molecular basis for high affinity interactions with C-2 derivatives of estradiol.J. Biol. Chem. 2002; 277 (12228253): 45219-4522510.1074/jbc.M207762200Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). The binding pocket was occupied by DVT in an orientation where vanillyl group A and the THF ring sit in the same position as a steroid, but the second vanillyl group B occupies a volume that is normally empty when a steroid is bound (Fig. 3A). This crystal structure implies that Ser-42 contributes significantly to the binding of DVT, as observed for all SHBG ligands (14Avvakumov G.V. Cherkasov A. Muller Y.A. Hammond G.L. Structural analyses of sex hormone-binding globulin reveal novel ligands and function.Mol. Cell Endocrinol. 2010; 316 (19748550): 13-2310.1016/j.mce.2009.09.005Crossref PubMed Scopus (62) Google Scholar), but this appears to be mediated through a water molecule that bridges Ser-42 and the oxygen of the THF ring (Fig. 3B). The other key interaction is a hydrogen bond between Asp-65 and the hydroxyl group of vanillyl group A that appears to also hold the ligand in place. A less significant contribution to binding is implied for Arg-135, which borders the flexible lidlike loop that is disordered (residues Leu-131–Ser-133) when DVT is bound (Fig. 2A). Although Arg-135 reaches into the binding pocket, it is not clear if it hydrogen bonds directly with DVT; instead, it may stabilizes the contact with Asp-65 through an ionic interaction (Fig. 3B). The disordering of the loop is caused by DVT displacing Leu-131 and an outward movement and rearrangement of the loop that allows Arg-135 to stabilize DVT binding, and this contrasts with the corresponding role of Lys-134 in estradiol-bound structures (Fig. 3A). The positioning of Trp-84, a surface residue near the flexible loop region of SHBG, in the DVT-bound structure mirrors that in the estradiol-bound SHBG structure (9Grishkovskaya I. Avvakumov G.V. Hammond G.L. Catalano M.G. Muller Y.A. Steroid ligands bind human sex hormone-binding globulin in specific orientations and produce distinct changes in protein conformation.J. Biol. Chem. 2002; 277 (12065592): 32086-3209310.1074/jbc.M203999200Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The crystal structure of E176K SHBG in complex with IPI was determined at 1.71 Å and solved by molecular replacement with the SHBG E176K estradiol-bound structure (Fig. 4). No major changes to the globular structure of the protein were noted, whereas structural variation occurred throughout the binding pocket. The binding of IPI was determined by three major interactions involving Ser-42, Phe-67, and Asp-65 (Fig. 3B). Serine 42 interacts with the nitrogen of the imidazole portion of IPI through a hydrogen bond of 2.7 Å. Residue Phe-67 π-π stacks with IPI through its phenyl group at a distance of 4 Å. Interestingly, this interaction forces Phe-67 into an alternate conformation as compared with when DVT or steroids are bound (Fig. 4A). This conformation of Phe-67 rearranges the hydrophobic binding pocket sufficiently to allow the perpendicular positioning of IPI relative to DHT (Fig. 4A), which leaves a large portion of the binding pocket empty in the IPI structure, and this space is filled by water molecules (Fig. 4B). Although Asp-65 is 6.9 Å away from IPI, two of these water molecules imply binding through a water molecule bridge ending at the nitrogen of the IPI indole group (Fig. 4B). The coordination of these water molecules appears to be supported by Arg-135, Asn-82, and Ser-128. Other residues that are important in accommodating steroids are integral to the hydrophobic pocket, and include Met-107 and Met-139 (3Grishkovskaya I. Avvakumov G.V. Sklenar G. Dales D. Hammond G.L. Muller Y.A. Crystal structure of human sex hormone-binding globulin: Steroid transport by a laminin G-like domain.EMBO J. 2000; 19 (10675319): 504-51210.1093/emboj/19.4.504Crossref PubMed Scopus (131) Google Scholar). In the IPI structure, Met-107 is modeled with multiple conformations appearing to increase the hydrophobic area to accommodate the indole portion of IPI (Fig. 4B). Additionally, Leu-171 projects 1.0 Å further into the binding pocket when compared with when estradiol is present (Fig. 4A). As in the DVT-bound structure, IPI binding shifts the loop region above the steroid-binding site outwards, as a result of Leu-131 being displaced by the indole of IPI, and this also causes Lys-134 to move out of the binding pocket and Arg-135 to orient toward the binding pocket. In the IPI-bound structure, Trp-84 is positioned as it is when DHT is bound (PDB: 1D2S). Substitutions of specific residues within the N-terminal LG domain of SHBG were made to validate crystal structure predictions of the interactions between SHBG and DVT or IPI (Table 2). Surprisingly, a S42A substitution that reduces affinity for DHT (19Hong E.-J. Sahu B. Jänne O.A. Hammond G.L. Cytoplasmic accumulation of incompletely glycosylated SHBG enhances androgen action in proximal tubule epithelial cells.Mol. Endocrinol. 2011; 25 (21193555): 269-28110.1210/me.2010-0483Crossref PubMed Scopus (23) Google Scholar) resulted in an ∼5-fold increased relative binding affinity (RBA) for DVT when compared with that of DHT (Table 2). By contrast, a D65A substitution abolished DVT binding to SHBG, supporting the assumption that Asp-65 hydrogen bonds with the hydroxyl of vanillyl A. The binding of DVT was also negatively impacted by the substitution of Arg-135 with leucine. The N82A substitution resulted in an increase in the RBA of DVT, likely caused by a more optimal conformation of the methoxy of vanillyl group A, as it appears that Asn-82 does not contribute a significant hydrogen bond to this interaction. Substitutions of residues within the hydrophobic pocket, Met-107, Met-139, and Phe-67 by smaller amino acids, resulted in modestly increased RBA for DVT (Table 2).Table 2IPI and DVT competitive binding analysis of CHO expressed SHBG mutants of key binding interaction residues identified from the structure modelSHBG mutantDHT IC50 (nm)IPI IC50 (nm)DVT IC50 (nm)IPI (RBA%)DVT (RBA%)Wildtype4.7 ± 0.755 ± 72500 ± 3708.50.19D65A4.0 ± 1.8430 ± 160NCD0.93NCDM107V3.6 ± 1.07.6 ± 1.3690 ± 350470.52M139V4.6 ± 1.741 ± 27590 ± 370110.78F67A5.1 ± 1.6390 ± 240820 ± 3201.30.62R135L3.0 ± 0.630 ± 133100 ± 1600100.10N82A3.3 ± 1.223 ± 7310 ± 180141.1E176K14 ± 2150 ± 164200 ± 7909.30.33S42A42 ± 9NCD4100 ± 950NCD1.0 Open table in a new tab The most significant residues contributing to IPI binding from the crystal structure appear to be Asp-65, Ser-42, and Phe-67. A D65A substitution decreases the RBA of IPI (Table 2) that is unlikely to be because of a structural change in the binding site, but it supports the possibility that Asp-65 contributes a hydrogen bond through water molecules (Fig. 3A). The SHBG S42A mutant showed no detectable competitive binding for IPI, supporting the predicted hydrogen bond between IPI and Ser-42. Study of the SHBG R135L mutant indicates that this residue does not contribute significantly to the binding of IPI, as was also observed for the N82A substitution. However, substitution of hydrophobic residues in the steroid-binding pocket caused changes in the affinity of SHBG for IPI. The F67A substitution greatly reduced the RBA of SHBG for IPI, which was expected because of the predicted importance to binding of the π-π stacking interaction occurring between the phenyl groups of IPI and Phe-67. The SHBG M139V mutant did not change the RBA for IPI, whereas the M107V mutant appears to have an increased affinity for IPI. Given the multiple conformations of Met-107 in the IPI-bound SHBG structure, it is reasonable to assume that the substitution of this residue with a smaller hydrophobic residue likely allows the orientation of IPI to be adjusted allowing for other interactions to be optimized. The relative affinities of SHBG for DVT and IPI were 1.5 and 74% of that measured for testosterone, respectively (Fig. 5A). These competition curves also demonstrate that the affinity for DVT is considerably lower than that of estradiol (34.2%), whereas IPI binds with a higher affinity than estradiol and almost as well as testosterone (Fig. 4A). We also found that (−)3,4-dibenzyltetrahydrofuran, a compound lacking the hydroxyl and methoxy groups of DVT, binds SHBG very poorly with an IC50 value >10 μm. Saturation of the SHBG zinc-binding site within the flexible loop region that covers the steroid binding pocket was also tested to determine how this might influence the relative binding affinities for DVT and IPI (Fig. 5B). This demonstrated that the affinities of SHBG for DVT and IPI were both negatively impacted in the presence of high Zn2+ concentrations. Under these conditions, the SHBG affinity for testosterone is unchanged whereas its affinity for estradiol is markedly reduced (Fig. 5, A versus B), as reported previously (11Avvakumov G.V. Muller Y.A. Hammond G.L. Steroid-binding specificity of human sex hormone-binding globulin is influenced by occupancy of a zinc-binding site.J. Biol. Chem. 2000; 275 (10859323): 25920-2592510.1074/jbc.M004484200Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). In the presence of high Zn2+ concentrations, DVT showed no detectable binding at all, whereas the affinity of SHBG for IPI was markedly reduced (by ∼20-fold) when compared with that of testosterone (Fig. 5B), and this effect was greater than ∼5-fold reduction in the affinity for estradiol, relative to that of testosterone. The influence of DVT and IPI on the ligand-dependent interaction between SHBG and fibulin-2 was assessed in a GST:fibulin-2 pulldown assay followed by Western blotting. A negative control (Sepharose linked to GST alone) confirmed that the presence of the carboxyl-terminal region of fibulin-2 is required to pull down human SHBG from a diluted serum sample (Fig. 6A). As reported previously (20Ng K.-M. Catalano M.G. Pinós T. Selva D.M. Avvakumov G.V. Munell F. Hammond G.L. Evidence that fibulin family members contribute to the steroid-dependent extravascular sequestration of sex hormone-binding globulin.J. Biol. Chem. 2006; 281 (16601122): 15853-1586110.1074/jbc.M512370200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), the saturation of the SHBG-binding site by estradiol under these conditions increased the interaction between fibulin-2 and SHBG significantly (p = 0.007), when compared with that observed using unliganded SHBG (Fig. 6B). Importantly, this interaction was not influenced by the binding of DVT to SHBG, whereas IPI enhanced the SHBG interaction with fibulin-2 significantly (p = 0.0403), and to a similar extent as that observed when the SHBG-binding site was fully occupied by estradiol (Fig. 6B). Using a cell line that stably expresses the androgen receptor and a luciferase reporter gene under the control of an androgen response element, we measured how the competitive displacement of testosterone from SHBG in culture media by DVT or IPI influenced androgen responses. The results indicate that the addition of 10 nm SHBG alone to the culture medium has no effect in the absence of testosterone, nor did the same concentration of heat-inactivated SHBG (Fig. 7). In the absence of SHBG, a robust ∼15-fold increase in luciferase response was observed upon treatment with 10 nm testosterone, and the presence of heat-inactivated SHBG had no effect on this. By contrast, the presence of 10 nm SHBG almost completely suppressed the activity of the same concentration of testosterone. The addition of 1 μm DVT alone did not cause an androgen response, whereas the addition of 500 nm (p = 0.0001) and 1 μm DVT (p = 0.0002) both cause significant increases in androgen activity when incubated together with 10 nm SHBG and 10