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Site-specific cation binding mediates TATA binding protein-DNA interaction from a hyperthermophilic archaeon
S Bergqvist1, R O'Brien, J E Ladbury
1Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.
Biochemistry
|May 1, 2001
Summary
The TATA box binding protein (TBP) from the hyperthermophilic archaeon Pyrococcus woesei binds DNA differently than its mesophilic counterparts. This study identifies a specific glutamate residue crucial for cation binding, influencing TBP-DNA interaction thermodynamics.
Area of Science:
- Biochemistry
- Molecular Biology
- Structural Biology
Background:
- Pyrococcus woesei (Pw) is a hyperthermophilic archaeon thriving in high salt and temperature.
- Previous work showed Pw TATA box binding protein (TBP) binds DNA differently than mesophilic TBPs, with increased affinity at higher salt concentrations.
- Protein-DNA complex formation in Pw TBP involves water release and ion uptake, hypothesized to be cations.
Purpose of the Study:
- To test the hypothesis that cations are involved in Pw TBP-DNA complex formation.
- To identify specific residues responsible for cation coordination at the protein-DNA interface.
- To elucidate the thermodynamic implications of cation binding on Pw TBP-DNA interactions.
Main Methods:
- Site-directed mutagenesis of acidic residues at the Pw TBP-DNA interface.
- Isothermal titration calorimetry (ITC) to determine thermodynamic parameters of mutant and wild-type TBP-DNA complex formation.
- Comparison of thermodynamic profiles to assess the role of specific residues in cation binding and overall affinity.
Main Results:
- Mutagenesis of a specific glutamate residue (E12) at the binding interface reduced cation uptake by one.
- This glutamate (E12) was confirmed to be directly involved in cation binding.
- Substitution of a proximal acidic residue (D101) did not affect cation uptake, highlighting the importance of residue location.
- Removal of the cation binding site enhanced binding entropy, particularly at low salt concentrations.
- The Pw TBP-DNA interaction affinity increased at low salt concentrations upon removal of the cation binding site.
Conclusions:
- A specific glutamate residue (E12) in Pw TBP is essential for coordinating cations at the protein-DNA interface.
- Cation binding significantly influences the thermodynamics of Pw TBP-DNA complex formation, particularly the entropic contribution.
- The findings reveal a unique mechanism of DNA binding in hyperthermophilic archaea, adapted to extreme environments.