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A mutation study of the DNA binding domain of human papillomavirus type11 E2 protein
T Matsumoto1, N Nakashima, K Takase
1Department of Molecular Biology, National Institute of Agrobiological Resources, Ibaraki. mat@abr.affrc.go.jp
Abstract:
A site-specific mutation study was performed on the C-terminal domain, containing a cloned DNA binding region, of the human papillomavirus type11 (HPV11) E2 protein to determine the specific properties of residues directly involved in the DNA binding. The effect of a point mutations on the DNA binding was assessed by means of a gel mobility shift assay. The mutagenesis was concentrated on the residues in the third helix from the N-terminal, that is known as the "recognition helix," in the crystal structure of the bovine papillomavirus (BPV) E2 protein. Most point mutations caused a great decrease in the DNA binding activity. The leucine repeat in the DNA binding region was proved not to be a leucine prerequisite, as the leucines could be substituted by valine without significant loss of the DNA binding ability. Substitution of Leu for Glu caused a significant decrease in the DNA binding, indicating that the hydrophobicity of the residue at this position is important. The results suggest that the individual contribution of each amino acid residue in the DNA binding region is essential for the DNA binding.
Insights
This study investigated mutations in the human papillomavirus type 11 (HPV11) E2 protein's DNA binding region. Results show most mutations decrease DNA binding, highlighting the essential role of individual amino acids in this process.
Area of Science:
- Molecular Biology
- Virology
- Protein-DNA Interactions
Background:
- The human papillomavirus type 11 (HPV11) E2 protein plays a crucial role in viral DNA replication and transcription.
- Understanding the DNA binding domain of the E2 protein is essential for deciphering viral regulatory mechanisms.
Purpose of the Study:
- To elucidate the specific roles of amino acid residues within the HPV11 E2 protein's DNA binding region.
- To identify key residues critical for mediating specific DNA interactions.
Main Methods:
- Site-specific mutagenesis was employed to alter residues in the C-terminal DNA binding domain of the HPV11 E2 protein.
- Gel mobility shift assays were utilized to quantitatively assess the impact of mutations on DNA binding affinity.
- Mutagenesis focused on the 'recognition helix' identified in related papillomavirus E2 structures.
Main Results:
- The majority of point mutations introduced into the E2 DNA binding region significantly reduced DNA binding activity.
- Leucine residues within the leucine repeat motif are not strictly required, as substitution with valine retained binding ability.
- Replacing leucine with glutamic acid drastically decreased DNA binding, emphasizing the importance of hydrophobicity at this position.
Conclusions:
- Each amino acid residue within the E2 DNA binding region contributes uniquely and essentially to DNA binding.
- The study provides critical insights into the structure-function relationship of the HPV11 E2 protein's DNA binding domain.