Functional conformer of c-Myb DNA-binding domain revealed by variable temperature studies

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biophysics

Background:

• The c-Myb protein's DNA-binding domain (R2R3) is crucial for gene regulation.
• Understanding its conformational dynamics under physiological conditions is essential for elucidating its function.

Purpose of the Study:

• To investigate the conformational fluctuations of the c-Myb R2R3 domain at physiological temperatures.
• To determine the role of a cavity in repeat 2 on protein dynamics and DNA-binding.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy, including 1D (1)H and 2D (15)N/(1)H spectra.
• Thermal denaturation studies to assess global unfolding transitions.
• Analysis of wild-type and cavity-filled mutant (V103L) proteins.

Main Results:

• A global unfolding transition was observed between 30-70 °C (Tm ≈ 50 °C).
• NMR data revealed local conformational fluctuations at physiological temperature (37 °C).
• The cavity in R2 significantly enhanced local fluctuations in the wild-type protein, an effect reduced in the V103L mutant.

Conclusions:

• The R2R3 domain exists in a dynamic equilibrium of conformers at 37 °C, with a more flexible conformer likely facilitating DNA binding.
• The cavity in R2 represents an evolutionary strategy to achieve optimal conformational flexibility for DNA interaction.
• These findings support the excited-state conformer model for protein function.