Effect of a salt-bridge between inter-repeats on the 3D structure of the c-Myb DNA-binding domain revealed by thermodynamic analysis

Abstract

Structure folding dictates protein functions. Recent advances in structural analysis such as NMR and X-ray crystallography enabled to determine protein structures at high resolution, which helps understanding of protein folding and recognition mechanisms. Additionally, solution thermodynamic analysis provides useful information to visualize the real behavior of proteins because they largely fluctuate in solution. In this study, we analyzed the effect of a putative salt-bridge between His-137 and Asp-178 in the c-Myb DNA-binding domain on the thermodynamics of its folding and motion. The minimum unit for specific DNA-binding of c-Myb consists of the two repeats, R2 and R3. The residues, His-137 and Asp-178, are located in R2 and R3, respectively, and are spatial proximity, possibly forming a salt-bridge as revealed by a previous crystal structure analysis. D178N mutation caused slight changes in the tertiary structure of R2R3. The thermal stability of the D178N mutant in the absence of DNA was much lower than that of wild-type R2R3. The decrease in stability was due to unfavorable enthalpy change, partially compensated by the favorable entropy change. The largely decreased enthalpy change indicated that the disruption of the salt-bridge weakens the overall intramolecular interactions in the folded state. Additionally, the increased entropy change indicated that the dynamics of the folded state increase upon mutation. In contrast, the D178N mutation slightly affected the thermal stability of the DNA-bound state, indicating that the salt-bridge is required for proper folding of R2R3 in the DNA-free state.