Abstract
Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 (hERR2) belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers. A carboxyl-terminal extension (CTE) to the zinc-finger domain has been implicated to be responsible for determining the stoichiometry of binding by a nuclear receptor to its response element. To better understand the mechanism by which DNA specificity is achieved, the solution structure of the DNA-binding domain of hERR2 (residues 96-194) consisting of the two putative zinc fingers and the requisite 26-amino acid CTE was analyzed by multidimensional heteronuclear magnetic resonance spectroscopy. The highly conserved zinc-finger region (residues 103-168) has a fold similar to those reported for steroid and retinoid receptors, with two helices that originate from the carboxyl-terminal ends of the two zinc fingers and that pack together orthogonally, forming a hydrophobic core. The CTE element of hERR2 is unstructured and highly flexible, exhibiting nearly random coil chemical shifts, extreme sensitivity of the backbone amide protons to solvent presaturation, and reduced heteronuclear (1H-15N) nuclear Overhauser effect values. This is in contrast to the dimer-binding retinoid X and thyroid hormone receptors, where, in each case, a helix has been observed within the CTE. The implications of this property of the hERR2 CTE are discussed.
Highlights
Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers
NMR Assignments of the Wild-type human estrogen related receptor-2 (hERR2) DNA-binding domain (DBD)—NMR studies were carried out on the recombinant DBD peptide, which contains the putative 66-amino acid (ZnCys4)2-type zincfinger domain and the requisite 26-amino acid carboxyl-terminal extension (CTE) for high affinity monomer binding to the extended half-site sequence [22, 45]. 87% of the backbone protons, 53% of the side-chain protons, and 86% of the backbone nitrogens have been assigned for Leu-102–Arg-174
We have constructed a recombinant form of the DBD of hERR2 and shown that it binds two zinc(II) ions/ polypeptide
Summary
Unlike steroid and retinoid receptors, which associate with DNA as dimers, human estrogen related receptor-2 (hERR2) belongs to a growing subclass of nuclear hormone receptors that bind DNA with high affinity as monomers. There is currently no structural data available on monomer-binding nuclear hormone receptors, several functional/mutagenesis studies have identified a carboxyl-terminal extension (CTE) of the zinc-finger modules as a second structural determinant that contributes to DNA binding.
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