Steroid-binding Specificity of Human Sex Hormone-binding Globulin Is Influenced by Occupancy of a Zinc-binding Site

Abstract

One calcium-binding site (site I) and a second poorly defined metal-binding site (site II) have been observed previously within the amino-terminal laminin G-like domain (G domain) of human sex hormone-binding globulin (SHBG). By soaking crystals of this structure in 2.5 mM ZnCl 2 , site II and a new metal-binding site ( Plasma sex hormone-binding globulin (SHBG)1 is a homodimeric glycoprotein produced by hepatocytes (1). It transports sex steroids in the blood and regulates their access to target cells (1, 2). The transcription unit responsible for plasma SHBG production by the liver is also expressed in rat Sertoli cells (3), and the protein product is known as the androgenbinding protein (ABP). Testicular ABP has exactly the same primary structure and steroid-binding properties as plasma SHBG and is thought to control androgen-dependent sperm maturation in the male reproductive tract (1, 3). Human SHBG and rat ABP comprise two laminin G-like domains (4) and share sequence similarity with the carboxyl-terminal regions of numerous extracellular proteins with diverse functions, including the blood coagulation co-factor, protein S; the growth factor, Gas 6; and several matrix-associated proteins (4). There is reason to suspect that the structural relationship between these proteins is functionally significant because their SHBGlike domains comprise binding sites for several members of an orphan tyrosine-kinase receptor superfamily (5). In addition, several groups have advanced the hypothesis that SHBG interacts with a plasma membrane receptor that contributes to steroid signaling pathways (6), but the biological impact of these interactions remains obscure.Studies of human and rabbit SHBG with the luminescent lanthanide, terbium, have indicated that each dimer of SHBG contains four metal-binding sites (7). At least one of these sites was assumed to bind calcium because calcium ions stabilize the steroid-binding activity of the purified protein (8) and enhance SHBG dimer formation (9). A calcium-binding site was recently located within the amino-terminal laminin G-like domain (G domain) of human SHBG, which was complexed with steroid ligand, but it is at least 20 Å from the closest steroid atom and is not close to the proposed dimer interface (10). In addition, a strong difference density peak was observed at the end of strand ␤6 in this crystal structure but was unsatisfactorily modeled as another calcium-binding site because of multiple conformations of the coordinating side chains (10).The location of this second metal-binding site was intriguing because it lies in the vicinity of a disordered region in the crystal structure of the amino-terminal G domain of human SHBG, which loops over the steroid-binding site (10). Moreover, amino acids within this particular region of human SHBG can be affinity labeled with reactive groups attached to steroid ligands (11), and their substitution with residues in the corresponding position of rat ABP results in altered steroid-binding specificity (9). The possibility that the...

Steroid Ligands Bind Human Sex Hormone-binding Globulin in Specific Orientations and Produce Distinct Changes in Protein Conformation

“…Therefore, the hydrogen bond between the side chain of Asp 65 and the steroid appears of particular importance for the binding of estradiol. This might also explain why the presence of a zinc ion in this region of SHBG, which causes the side chain of Asp 65 to move away from the steroid, only affects the binding of estradiol (14). By contrast, the binding affinities of the N82A variant for both androgen and estradiol were normal.…”

“…These include a confirmation of the proposed dimer interface and the demonstration that each SHBG monomer contains a functional steroid-binding site (12) rather than a single binding site at the homodimer interface (13). Our crystal structure studies have also led to the discovery that SHBG is a zinc-binding protein and that occupancy of a zinc-binding site alters the specificity of steroid binding (14). This zinc-binding site lies within a loop region that was disordered in our original crystal structure (8): a region of particular interest because it is poorly conserved between species (15) and amino acid substitutions within it influence steroid-binding specificity (11).…”

“…3 H]DHT (42 Ci/mmol; PerkinElmer Life Sciences) as the labeled ligand was used to determine the steroid binding capacity, affinity, and specificity of purified human SHBG (14) or recombinant wild-type human SHBG and human SHBG variants in Chinese hamster ovary cell media after concentration and dialysis, as described above. In all cases, the amount of SHBG in samples for analysis was adjusted so that DHT-binding capacities were within 10% of each other.…”

“…As noted previously (11), substitution of His 136 with Gln led to a specific reduction in the SHBG binding affinity for estradiol (Table III). However, the altered position of His 136 when a zinc-binding site in this region is occupied causes a marked and specific reduction in estradiol binding (14), and it is therefore possible that a glutamine at this position has an effect on the conformation of the loop segment covering the steroid-binding site. Among other residues in this loop segment, previous reports using affinity labeling techniques have shown that Lys 134 is located close to the steroid ligand (10), and when this residue was substituted with histidine it again led to a small but specific reduction in the binding affinity for estradiol (11).…”

A Human Sex Hormone-binding Globulin Isoform Accumulates in the Acrosome during Spermatogenesis

“…Immunohistochemistry-The anti-human SHBG antibodies for immunohistochemistry were purified from a rabbit antiserum by immunoaffinity chromatography using an N-hydroxysuccinimide-activated HiTrap column coupled to purified human SHBG (16), according to instructions provided by Amersham Biosciences. Testes from perfused mice (see above) were further fixed in 4% paraformaldehyde at 4°C for 24 h, subsequently dehydrated with a series of ethanol solutions, and embedded in paraffin.…”