Metal-dependent folding and stability of nuclear hormone receptor DNA-binding domains.

Journal article

The nuclear/hormone receptors are an extensive family of ligand-activated transcription factors that recognise DNA targets through a highly conserved, structurally autonomous DNA-binding domain. The compact structure of the DNA-binding domain is supported by two zinc ions, each of which is co-ordinated by the tetrahedral arrangement of thiol groups from four cysteine residues. Metal binding is expected to be linked with deprotonation of the co-ordinating thiol groups and folding of the polypeptide. Using a variety of biophysical approaches, we characterise these linked equilibria for the isolated DNA-binding domains (DBD) of the receptors for estrogen and glucocorticoid. Mass spectrometry and equilibrium denaturation indicate that, near neutral pH, approximately four of the eight co-ordinating thiol groups release protons with zinc uptake, in agreement with the expected pK(a) change for the -SH group in the presence of the metal. Mass spectrometry reveals that the protein charge distribution changes with the uptake of zinc and that metal binding is co-operative. The co-operativity is consistent with observations from equilibrium denaturation, which indicate that the folding event is a two-state process. A crucial residue that stabilises the equilibrium structure of the DBD fold itself is a cysteine residue situated in the hydrophobic core of all known nuclear hormone receptors (but not involved in metal binding): it appears to be conserved absolutely for its unique combination of size and hydrophobicity. Stabilisation of the DBDs could be achieved by truncating the flexible, basic termini, suggesting that like-charge clusters may have deleterious effects on protein folds. While the metal-free apo protein and the chemically denatured state have little defined secondary structure, these states were expanded only partially in comparison with the native structure, according to data from small-angle X-ray scattering. The comparatively compact shapes of the denatured and apo forms may explain, in part, the marginal stability of the native fold.

Authors: Low, L; Hernández, H; Robinson, C; O'Brien, R; Grossmann, J

• Publication status: Published
• Journal: Journal of molecular biology
• Volume: 319
• Issue: 1
• Pages: 87-106
• Publication date: 2002-05-01
• DOI: 10.1016/s0022-2836(02)00236-x
• EISSN: 1089-8638
• ISSN: 0022-2836
• Language: English
• Keywords: Receptors, Cytoplasmic and Nuclear; Amino Acid Sequence; Hydrogen-Ion Concentration; Cysteine; DNA-Binding Proteins; Protein Conformation; Protein Binding; Protein Structure, Tertiary; Molecular Weight; Calorimetry; Protein Folding; Apoproteins; Protein Denaturation; Zinc; Receptors, Estrogen; Spectrometry, Fluorescence; Models, Molecular; Spectrometry, Mass, Electrospray Ionization; DNA; X-Ray Diffraction; Receptors, Glucocorticoid; Circular Dichroism; Nuclear Magnetic Resonance, Biomolecular; Molecular Sequence Data; Mutation; Thermodynamics