The nuclear receptor superfamily is composed of ligand-dependent transcription factors that regulate a variety of cellular processes including metabolism, development, growth, differentiation, and reproduction. Nuclear receptors are modular proteins containing a conserved DNA binding domain (DBD), a C-terminal ligand binding domain (LBD), and a diverse amino-terminal region (1
). Transcriptional regulation by nuclear receptors primarily occurs through direct binding to specific hormone response elements (HREs) of target genes. The nuclear receptor superfamily can be subdivided based on distinct DNA binding mechanisms. Class I receptors for steroid hormones interact optimally as head-to-head homodimers with hexanucleotide HREs arranged as inverted repeats with a trinucleotide spacer (3N) between the half-sites (2
). There are two hexanucleotide HREs for the steroid receptors: AGGTCA is preferred by estrogen receptor (ER), as well as by the class II and orphan receptors, and AGAACA is preferred by the glucocorticoid receptor (GR) subfamily, which also includes androgen receptor (AR), mineralocorticoid receptor (MR), and progesterone receptor (PR). Class II receptors for the nonsteroidal hormones and dietary lipids such as retinoic acid receptor (RAR), thyroid hormone receptor (TR), vitamin D3 receptor (VDR), and peroxisome proliferator-activated receptor (PPAR), bind primarily as heterodimers together with retinoic acid X receptor (RXR) to HREs arranged as direct repeats with variable spacing between the half-sites (1
). Orphan receptors, for which physiological ligands are unidentified, interact through various configurations including monomer binding to extended HRE half-sites (1
The DNA binding regions of the nuclear receptors share a core DBD, consisting of two α
helices and two asymmetric zinc binding modules coordinated by eight conserved cysteine residues, followed by a less conserved region termed the C-terminal extension (CTE). The first helix of the core DBD inserts into the major groove of the HRE, making base-specific contacts, and helix 2 crosses over the top of helix 1 and the major groove leading into the CTE. The dimerization domain (D box) resides within the C-terminal zinc-binding module and is important for mediating DNA binding-dependent dimerization of receptors (8
Unlike the core DBD, the CTE is not well conserved in sequence or structure. Structural studies of class II and orphan receptors have shown that the CTE, which has a highly variable length of up to 40 amino acids beyond the conserved Gly-Met sequence at the C terminus of the core DBD (see ), provides additional contacts with the DNA minor groove outside of the canonical HREs. The CTEs of TR and VDR form a third α
-helix that projects across the minor groove to make extensive contacts with the phosphate backbone of spacer nucleotides. The CTEs of TR and VDR are also important for DNA-dependent homo- and heterodimerization, as well as for proper spacing of receptor subunits between direct-repeat response-element half-sites (12
). The CTEs of the orphan receptors [RevErb, nerve growth factor-I-β
, liver receptor homolog-1, and estrogen-related receptor-2 (ERR2)] form an extended loop that occupies the minor groove of DNA sites flanking the HRE (8
). In addition to the extended loop the liver receptor homolog-1 DBD contains a non-DNA binding α
-helix termed the Ftz-F1 motif (19
). Conformational changes in the CTE have been observed in response to DNA binding. The RXR CTE is an alpha helix in solution but forms an extended loop conformation when bound to DNA, whereas the CTE of ERR2 is unstructured in solution and becomes structured when bound to DNA (9
). Biochemical and mutagenesis studies have also shown that the CTE is required for high affinity DNA binding by class II and orphan nuclear receptors (5
). In particular, the CTE is important for the DNA binding of orphan receptors that interact as monomers with a half-site HRE. The CTE effectively extends the protein-DNA interface, increases the stability of monomer binding to HRE half-sites, and creates a preference for 5′ trinucleotide sequences flanking half-site HREs.
In contrast to class II and orphan receptors, much less is known about the steroid receptor CTEs. In previous studies, we showed that the CTE of ER and PR is required for interaction with the high mobility group protein-1 and -2 (HMGB-1/-2) that function as coregulatory proteins for all members of the steroid class of receptors by facilitating receptor binding to specific DNA target sites (25
). Similar to the orphan receptors, the CTE of ER and PR was also required for efficient binding of ER and PR to HRE half-sites (31
). However, structural studies of steroid receptors DBDs including GR, AR, and ER, either lacked sufficient CTE sequence in the protein expression constructs, or the CTE was disordered in the crystal structures (34
). Furthermore, there are no reports of structures of a PR-DBD/DNA complex.
To understand the role of the CTE in PR-DNA binding, we determined the crystal structure of the PR DBD including the CTE bound to consensus inverted-repeat progesterone response elements (PREs). Arg637 and Lys638 of the CTE contact the DNA minor groove at sites flanking the PREs. Alanine substitutions of Arg637 and Lys638 reduced binding affinity of PR to the inverted repeat PREs and abrogated binding of PR to a half-site PRE. Other novel structural features of the PR-DBD DNA complex included interactions of the subunits with the PRE consensus sequence bound by the opposite subunit, which coincided with a narrow minor groove in the 3N spacer DNA. These studies further our understanding of steroid receptor-DNA recognition mechanism and suggest that minor groove DNA interactions outside the HREs contribute to binding affinity and sequence selectivity.