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

J Mol Biol. 2002 May 24;319(1):87-106. doi: 10.1016/S0022-2836(02)00236-X.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Apoproteins / chemistry
  • Apoproteins / genetics
  • Apoproteins / metabolism
  • Calorimetry
  • Circular Dichroism
  • Cysteine / genetics
  • Cysteine / metabolism
  • DNA / genetics
  • DNA / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Molecular Sequence Data
  • Molecular Weight
  • Mutation
  • Nuclear Magnetic Resonance, Biomolecular
  • Protein Binding
  • Protein Conformation
  • Protein Denaturation / drug effects
  • Protein Folding*
  • Protein Structure, Tertiary / drug effects
  • Receptors, Cytoplasmic and Nuclear / chemistry*
  • Receptors, Cytoplasmic and Nuclear / genetics
  • Receptors, Cytoplasmic and Nuclear / metabolism*
  • Receptors, Estrogen / chemistry
  • Receptors, Estrogen / genetics
  • Receptors, Estrogen / metabolism
  • Receptors, Glucocorticoid / chemistry
  • Receptors, Glucocorticoid / genetics
  • Receptors, Glucocorticoid / metabolism
  • Spectrometry, Fluorescence
  • Spectrometry, Mass, Electrospray Ionization
  • Thermodynamics
  • X-Ray Diffraction
  • Zinc / metabolism
  • Zinc / pharmacology*

Substances

  • Apoproteins
  • DNA-Binding Proteins
  • Receptors, Cytoplasmic and Nuclear
  • Receptors, Estrogen
  • Receptors, Glucocorticoid
  • DNA
  • Zinc
  • Cysteine