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

Abstract

Sex hormone-binding globulin (SHBG)1 is the major plasma transport protein for biologically active sex steroids in a wide range of vertebrate species, and changes in the plasma concentrations of SHBG play a key role in determining the plasma half-lives of its steroid ligands and their access to target tissues (1). The affinity and specificity of SHBG for steroid ligands varies between species, and human SHBG has a high affinity for both testosterone and 17␤-estradiol (1, 2) as well as androgen and estrogen metabolites that exert biological effects mediated by nongenomic mechanisms (3). Human SHBG also binds synthetic progestins used in oral contraceptives with close to nanomolar affinities (4) and interacts with several natural and synthetic xenobiotics, albeit with lower affinity (5). All of these natural and synthetic ligands compete with each other for binding to SHBG, but nothing is known about how they are coordinated within its steroid-binding site.The realization that the steroid-binding and dimerization domains of human SHBG are located within its amino-terminal laminin G-like (LG) domain (6) prompted us to crystallize this region of the molecule (7) in a complex with 5␣-dihydrotestosterone (DHT). This allowed us to visualize the structure of the steroid-binding site (8) and to fully appreciate the results of earlier affinity labeling experiments (9, 10) as well as biochemical studies of recombinant human SHBG variants containing specific amino acid substitutions (11). Subsequent analysis of the amino-terminal LG domain of SHBG has revealed several other features of the molecule that add new dimensions to our understanding of its structure and function. 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). Resolution of the structure of this extended loop region has recently helped us to understand how zinc influences the conformation and steroidbinding specificity of human SHBG (16).Because different steroids compete for a single binding site within the SHBG molecule, it has always been assumed that they enter the site in the same orientation. However, there are reasons to believe that particular steroid ligands are coordinated differently within the binding site and may cause specific conformational changes in protein structure. For instance,

Evidence That Fibulin Family Members Contribute to the Steroid-dependent Extravascular Sequestration of Sex Hormone-binding Globulin

“…Our data clearly imply that the fibulin-binding site on SHBG is located within its LG-4 domain and probably involves regions that are configured differently in the presence of androgens versus estrogens, such as the flexible loop located over the steroid-binding site (7,8). Because the carboxyl-terminal regions of fibulin-1D and fibulin-2 both failed to recognize the SHBG LG-5 domain in yeast two-hybrid experiments, we were surprised that the elimination of N-linked glycosylation sites within the LG-5 domain of SHBG significantly enhanced its ability to interact with the carboxyl-terminal regions of fibulin-1D and fibulin-2 in the GST pull-down assays.…”

“…High resolution crystal structure analyses have shown that each subunit of the human SHBG homodimer contains a high affinity binding site for both testosterone and estradiol within its amino-terminal laminin G-like (LG-4) domain (4 -6) and that androgens and estrogens are oriented differently within this site (7). Moreover, occupancy of the steroid-binding site by different types of steroids influences the conformation of the LG-4 domain (6), especially in relation to a flexible loop structure that covers the steroid-binding pocket (8).…”

Curariform Antagonists Bind in Different Orientations to the Nicotinic Receptor Ligand Binding Domain

“…The origin of the different orientations was ascribed to differences in the x-ray structures of the two antagonists, likely to be present in solution (26), that allow rearrangement of the positively charged ligands in the aromatic-rich binding site. Distinct docking orientations have also been observed for different steroids bound to sex hormone binding globulin, as determined by x-ray crystallography and confirmed by mutagenesis together with measurements of binding affinity (28). C19 androgens and estradiol were found to bind in opposite orientations where the A and D rings of the two steroids switch places within the binding pocket and the planes of the scaffolds flip by 180 o .…”

Sex hormone-binding globulin gene expression in the liver: Drugs and the metabolic syndrome

“…Circulating SHBG is a homodimeric plasma glycoprotein with a single steroid-binding site within each subunit. The mechanism of steroid ligand interactions with the two potential steroid-binding sites of the SHBG homodimer (Grishkovskaya et al, 2000), and the identification of the structural domains that directly or indirectly account for the steroid-binding specificity of human SHBG have been resolved at the atomic level by crystal structure analysis (Grishkovskaya et al, 2002), although some questions remain (see review by Hammond et al this issue).…”