Investigating urea-induced denaturation of human aurora kinase B: Integrated spectroscopic and computational approaches

Aurora kinase B (AURKB) is an essential component of the chromosome passenger complex and plays a key role in cellular mitosis. Given that abnormal mitosis contributes to cancer pathogenesis, AURKB has emerged as a promising target for anti-cancer drug development. To explore the biophysical characteristics of AURKB, urea-induced denaturation study was performed at pH 7.5 and 25 °C using far-UV, circular dichroism, and tryptophan fluorescence spectroscopy studies. The spectroscopic probes were employed in determining the stability parameters, including ΔG⁰_D (Gibbs free energy change in the absence of urea), C_m (the midpoint of the denaturation curve, representing the molar urea concentration at which ΔG_D=0), and m (the slope of the ΔG_D versus [urea] plot) which were found to be 4.16 ± 0.3 kcal mol^-1, 2.84 ± 0.06 M and 1.46 ± 0.10 kcal mol^-1 M^-1 respectively. The normalized denaturation curves of these optical properties suggest that AURKB undergoes a two-stage denaturation process, with structural unfolding beginning at 2.0 M urea and complete loss of structure at 4.0 M urea. Also, increasing urea exposure reduced the catalytic activity of recombinant AURKB. Additionally, 200 ns molecular dynamics (MD) simulations were performed to gain atomistic insights into the structural changes in AURKB under increasing urea concentrations. The findings of MD simulations aligned with the spectroscopic data, revealing a clear pattern of AURKB unfolding upon an increase in urea concentration. Together, the spectroscopic and MD simulation findings provide a comprehensive understanding of the urea-induced unfolding mechanism and conformational dynamics of AURKB, offering valuable information for designing targeted AURKB inhibitors for therapeutic applications.