title: Metal-dependent folding and stability of nuclear hormone receptor DNA-binding domains.
abstract: 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.affiliation: Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK
authors.forename: Lieh Yoon
authors.initials: LY
authors.lastname: Low
authors.forename: Helena
authors.initials: H
authors.lastname: Hernández
authors.forename: Carol V
authors.initials: CV
authors.lastname: Robinson
authors.forename: Ronan
authors.initials: R
authors.lastname: O'Brien
authors.forename: J Günter
authors.initials: JG
authors.lastname: Grossmann
authors.forename: John E
authors.initials: JE
authors.lastname: Ladbury
authors.forename: Ben
authors.initials: B
authors.lastname: Luisi
mesh_terms: D000595:Amino Acid Sequence; D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002151:Calorimetry; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation / Q000187:drug effects; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000187:drug effects; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011960:Receptors, Estrogen / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011965:Receptors, Glucocorticoid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization; D013816:Thermodynamics; D014961:X-Ray Diffraction; D015032:Zinc / Q000378:metabolism / Q000494:pharmacology*
chemical_list: D001059:Apoproteins; D004268:DNA-Binding Proteins; D018160:Receptors, Cytoplasmic and Nuclear; D011960:Receptors, Estrogen; D011965:Receptors, Glucocorticoid; D004247:DNA; D015032:Zinc; D003545:Cysteine