i: E/F

Nuclear Localization Signal l-c

Nuclear Localization Signal

Fig.16.1 General stucture of nuclear hormone receptors.

The second subdomain of the NR DBD also contains a zinc-finger motif and an a-helix, termed Helix III. As with the zinc-finger motif in the first subdomain, a single zinc ion is tetrahedrically complexed to four highly conserved cysteine residues in the second subdomain (Luisi et al., 1991). The existence of two zinc-finger motifs in such close proximity allows these motifs to fold together, giving the NR DBD its characteristic tertiary structure. In this tertiary structure, Helix III is oriented perpendicular to Helix I. The second subdomain of the DBD contains one other important region, termed the "D-box". This region is composed of amino acids in the zinc-finger of the second subdomain, and is partly responsible for the dimerization of the NRs (Aranda and Pascual, 2001; Kumar and Thompson, 1999).

C: The Hinge Domain: Domain D

The hinge domain is not well conserved among members of the NR superfamily. This domain serves as a linker between the DBD and the ligand binding domain (LBD, Domain E), and allows for rotation between these two domains. The hinge domain also contains a nuclear localization signal which is critical for the nuclear translocation of NRs (Aranda and Pascual, 2001).

D: The Ligand Binding Domain and C-Terminal Extension: Domain E/F

The ligand binding domain (LBD) of NRs is a multifunctional domain which contains regions mediating binding of ligand, homo- and heterodimerization, and ligand-dependent transcriptional activity. The function of Domain F, by contrast, is not currently known. This domain is mentioned in general NR superfamily structure out of recognition for its existence in some members of the superfamily. However, given the general lack of understanding of its function, it will not be discussed further here (Aranda and Pascual, 2001).

Crystal structures of the LBD of multiple NRs have been solved and have demonstrated some level of similarity between LBDs from different NRs. Based on these studies, a canonical three-dimensional structure of NRs has been proposed (Moras and Gronemeyer, 1998). In this structure, the NR LBD is composed of a series of 12a -helical regions, termed helices 1-12, closely folded into a three-layer, anti-parallel helical "sandwich" (Bourguet et al., 1995). A highly conserved a-turn is situated between helices 5 and 6. In this conformation, a central core layer of three helices is packed between 2 additional layers of helices, creating a cavity which can accommodate a ligand (Aranda and Pascual, 2001).

Contacts between amino acids in the ligand binding pocket and the ligand can be extensive and varies among different receptors. In some NRs, the ligand binding pocket of the LBD is quite large, allowing the binding of several differently-sized ligands. In other NRs, a smaller ligand binding pocket accommodates only a single, specific ligand (Uppenberg et al., 1998). Regardless of the ligand pocket size, interaction between residues in the LBD and the ligand are hypothesized to result in a conformational change which may have important consequences for NR function (Aranda and Pascual, 2001).

Within the ligand binding domain are two important regions, a "signature motif' and an Activation Function-2 (AF-2) motif. The AF-2 motif, located on helix 12 of the LBD, is a region that has been shown to be essential for ligand-induced transcriptional activation. However, the AF-2 motif does not act alone in this capacity, and must interact with a region called the "signature motif' located on and between helices 3 and 4. It has been hypothesized that the conformational change induced by ligand binding to the LBD results in physical approximation between AF-2 and the signature motif. This results in the formation of a surface over the liganded NR which is capable of interacting with other protein mediators of transcription (Danielian et al., 1992; Mclnerney et al., 1996). This interaction is described in more detail below.

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