Carboxyl-terminal domain dimer interface mutant 434 repressors have altered dimerization and DNA binding...

Area of Science:

• Molecular Biology
• Protein-DNA Interactions
• Structural Biology

Background:

• The 434 repressor protein requires strong dimerization for specific DNA binding and tetramer formation.
• Dimerization is mediated by the carboxyl (C)-terminal domain of the repressor.

Purpose of the Study:

• To investigate the role of specific amino acid residues in the C-terminal domain of the 434 repressor in dimerization.
• To understand how C-terminal domain dimerization affects DNA binding affinity and specificity.

Main Methods:

• Computer modeling based on homologous protein crystal structures.
• Site-directed mutagenesis of the 434 repressor C-terminal domain.
• Gel filtration and crosslinking assays to assess dimerization.
• Analysis of repressor-operator DNA binding affinity.

Main Results:

• Amino acid residues K121, H139, D161, and N163 were identified as critical for dimerization strength and specificity.
• Mutant repressors' dimerization ability did not correlate with their DNA binding capabilities.
• Mutant proteins showed deficiencies in detecting sequence-dependent DNA structural variations.

Conclusions:

• The structural integrity of the C-terminal domain dimer interface is essential for proper DNA binding.
• Mutations in the dimer interface alter DNA-protein complex structure, impacting DNA recognition.
• Proper orientation of the DNA-binding N-terminal domain relies on the C-terminal domain's dimerization interface.