Tet repressor mutants with altered effector binding and allostery

FEBS J. 2005 Sep;272(17):4487-96. doi: 10.1111/j.1742-4658.2005.04868.x.

Abstract

To learn about the correlation between allostery and ligand binding of the Tet repressor (TetR) we analyzed the effect of mutations in the DNA reading head-core interface on the effector specific TetR(i2) variant. The same mutations in these subdomains can lead to completely different activities, e.g. the V99G exchange in the wild-type leads to corepression by 4-ddma-atc without altering DNA binding. However, in TetR(i2) it leads to 4-ddma-atc dependent repression in combination with reduced DNA binding in the absence of effector. The thermodynamic analysis of effector binding revealed decreased affinities and positive cooperativity. Thus, mutations in this interface can influence DNA binding as well as effector binding, albeit both ligand binding sites are not in direct contact to these altered residues. This finding represents a novel communication mode of TetR. Thus, allostery may not only operate by the structural change proposed on the basis of the crystal structures.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Allosteric Regulation
  • Amino Acid Substitution
  • Base Sequence
  • Binding Sites
  • DNA, Bacterial / genetics
  • DNA, Bacterial / metabolism
  • Escherichia coli / drug effects
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Genes, Bacterial
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Protein Conformation
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Repressor Proteins / chemistry
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism*
  • Tetracycline Resistance / genetics
  • Tetracyclines / chemistry
  • Tetracyclines / pharmacology
  • Thermodynamics

Substances

  • DNA, Bacterial
  • Recombinant Proteins
  • Repressor Proteins
  • Tetracyclines
  • tetracycline resistance-encoding transposon repressor protein
  • 4-epianhydrotetracycline