Crystal structure of human sex hormone-binding globulin: steroid transport by a laminin G-like domain

Abstract

Human sex hormone-binding globulin (SHBG) transports sex steroids in blood and regulates their access to target tissues. In biological fluids, SHBG exists as a homodimer and each monomer comprises two laminin G-like domains (G domains). The crystal structure of the N-terminal G domain of SHBG in complex with 5alpha-dihydrotestosterone at 1.55 A resolution reveals both the architecture of the steroid-binding site and the quaternary structure of the dimer. We also show that G domains have jellyroll topology and are structurally related to pentraxin. In each SHBG monomer, the steroid intercalates into a hydrophobic pocket within the beta-sheet sandwich. The steroid and a 20 A distant calcium ion are not located at the dimer interface. Instead, two separate steroid-binding pockets and calcium-binding sites exist per dimer. The structure displays intriguing disorder for loop segment Pro130-Arg135. In all other jellyroll proteins, this loop is well ordered. If modelled accordingly, it covers the steroid-binding site and could thereby regulate access of ligands to the binding pocket.

Fig. 1. Modular architecture of SHBG and its homologues GAS6, protein S and laminin. The SHBG-like domain consists of a tandem repeat of laminin G–like domains. GAS6 and protein S both contain an N–terminal GLA domain and four EGF-like domains. The domain architecture of the laminin α–chain is complex (Beck et al., 1990). Within each protein, the G domains share little sequence identity, and the fragment of SHBG analysed here, namely the N–terminal G domain, is homologous to the N–terminal G domain of GAS6, protein S and the fourth G domain of the laminin α–chain.

Fig. 2. The G domain fold in SHBG. (A and B) Ribbon representation of the N–terminal domain of SHBG in two orthogonal orientations. The β–strands of the two sheets forming a β–sandwich are coloured in yellow and blue, respectively. The steroid 5α–DHT is shown in a ball and stick representation. The segment 130–135, which is expected to loop over the steroid-binding pocket, is disordered and not visible in the electron density. The calcium ion is shown as a green dot (figures generated with MOLSCRIPT and RASTER3D; Kraulis, 1991; Merritt and Murphy, 1994). (C) Topology diagram of the β–strands. Triangles pointing upwards denote β–strands pointing out of the paper plane. The conserved disulfide bond between residues 164 and 188 is indicated as a dashed line.

Fig. 3. (A) Ribbon representation of the human SHBG dimer coloured according to the atomic displacement factors (temperature factors) and model for the packing of the C–terminal G domains (in grey). The displacement factors range from 12 (dark blue) to 55 Ų (red). (B) Stereo representation of the steroid-binding pocket in an orientation identical to that in Figure 2B. All side chains that are in contact with the steroid are displayed. (C) Chemical structure and atom numbering in 5α–DHT. In testosterone, a double bond is present between atoms C4 and C5. In oestradiol, ring A is aromatic, C19 is missing and the carbonyl oxygen at C3 is replaced by a hydroxyl group.

Fig. 4. Multiple sequence alignment of the first G domain in the SHBG family. Sequence identities between human and rat SHBG (hSHBG, rSHBG), human protein S (hProt S), human GAS6 (hGAS6) and G domain 4 of the human laminin α–chain (hLamG4) are 71, 26, 25 and 19%, respectively. Secondary structure elements of SHBG are shown as grey boxes; residues involved in steroid binding (red), in the dimerization interface (blue), in calcium binding (yellow) and in disulfide bridge formation (green) are highlighted.