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A novel class of winged helix-turn-helix protein: the DNA-binding domain of Mu transposase
R T Clubb1, J G Omichinski, H Savilahti
1Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520.
Background:
Mu transposase (MuA) is a multidomain protein encoded by the bacteriophage Mu genome. It is responsible for translocation of the Mu genome, which is the largest and most efficient transposon known. While the various domains of MuA have been delineated by means of biochemical methods, no data have been obtained to date relating to its tertiary structure.
Results:
We have solved the three-dimensional solution structure of the DNA-binding domain (residues 1-76; MuA76) of MuA by multidimensional heteronuclear NMR spectroscopy. The structure consists of a three-membered alpha-helical bundle buttressed by a three-stranded antiparallel beta-sheet. Helices H1 and H2 and the seven-residue turn connecting them comprise a helix-turn-helix (HTH) motif. In addition, there is a long nine-residue flexible loop or wing connecting strands B2 and B3 of the sheet. NMR studies of MuA76 complexed with a consensus DNA site from the internal activation region of the Mu genome indicate that the wing and the second helix of the HTH motif are significantly perturbed upon DNA binding.
Conclusions:
While the general appearance of the DNA-binding domain of MuA76 is similar to that of other winged HTH proteins, the connectivity of the secondary structure elements is permuted. Hence, the fold of MuA76 represents a novel class of winged HTH DNA-binding domain.
Insights
Researchers determined the three-dimensional structure of the Mu transposase (MuA) DNA-binding domain using NMR. This novel winged helix-turn-helix (HTH) fold reveals insights into the mechanism of this efficient transposon.
Area of Science:
- Structural Biology
- Molecular Biology
- Biochemistry
Background:
- Mu transposase (MuA) is essential for bacteriophage Mu genome translocation.
- MuA is part of the largest and most efficient known transposon.
- The tertiary structure of MuA remained previously uncharacterized.
Purpose of the Study:
- To elucidate the three-dimensional structure of the MuA DNA-binding domain.
- To investigate the structural basis of MuA's DNA binding and translocation activity.
Main Methods:
- Multidimensional heteronuclear NMR spectroscopy was employed.
- The structure of the DNA-binding domain (MuA76) was solved in solution.
- NMR was used to study MuA76 in complex with a consensus DNA site.
Main Results:
- The three-dimensional solution structure of MuA76 was determined.
- MuA76 features a novel winged helix-turn-helix (HTH) motif with a permuted secondary structure element connectivity.
- DNA binding significantly perturbs the HTH motif and the flexible wing region.
Conclusions:
- The fold of MuA76 represents a new class of winged HTH DNA-binding domains.
- This structural determination provides a foundation for understanding MuA's transposition mechanism.
- The findings highlight the unique structural adaptations of MuA for efficient genome manipulation.