Crystal structure, folding, and operator binding of the hyperstable Arc repressor mutant PL8

Area of Science:

• Molecular Biology
• Protein Structure and Function
• Biochemistry

Background:

• Arc repressor is a dimeric DNA-binding protein from the ribbon-helix-helix transcription factor family.
• Protein stability and DNA binding affinity are critical for transcription factor function.

Purpose of the Study:

• To investigate the structural and functional consequences of a specific mutation (Pro8 to Leucine) in Arc repressor.
• To understand how enhanced protein stability affects DNA binding affinity and folding kinetics.

Main Methods:

• X-ray crystallography was used to determine the structure of the PL8 mutant dimer at 2.4-A resolution.
• Protein stability was assessed by comparing refolding and unfolding kinetics of the mutant and wild-type Arc.
• DNA binding affinity was evaluated through operator binding assays.

Main Results:

• The PL8 mutant exhibited increased stability (2.5 kcal/mol of dimer) due to additional interstrand hydrogen bonds in the beta-sheet, increasing total bonds from six to eight.
• Despite enhanced stability, the PL8 mutant showed significantly reduced DNA binding affinity compared to wild-type Arc.
• The mutation stabilizes the native state by decreasing the unfolding rate, indicating the beta-sheet forms post-rate-limiting folding step.

Conclusions:

• The Pro8 to Leucine mutation in Arc repressor enhances protein stability by reinforcing the beta-sheet structure.
• The trade-off between stability and DNA binding suggests that the structural changes preventing optimal operator contacts are responsible for reduced affinity.
• This study provides insights into the relationship between protein structure, stability, and DNA-binding function in transcription factors.