Mechanistic insights into the urea-induced denaturation of kinase domain of human integrin linked kinase

Integrin-linked kinase (ILK), a ubiquitously expressed intracellular Ser/Thr protein kinase, plays a major role in the oncogenesis and tumour progression. The conformational stability and unfolding of kinase domain of ILK (ILK_193–446) was examined in the presence of increasing concentrations of urea. The stability parameters of the urea-induced denaturation were measured by monitoring changes in [θ]₂₂₂ (mean residue ellipticity at 222 nm), difference absorption coefficient at 292 nm (Δε₂₉₂) and intrinsic fluorescence emission intensity at pH 7.5 and 25 ± 0.1 °C. The urea-induced denaturation was found to be reversible. The protein unfolding transition occurred in the urea concentration range 3.0–7.0 M. A coincidence of normalized denaturation curves of optical properties ([θ]₂₂₂, Δε₂₉₂ and λ_max, the wavelength of maximum emission intensity) suggested that urea-induced denaturation of kinase domain of ILK is a two-state process. We further performed molecular dynamics simulation for 100 ns to see the effect of urea on structural stability of kinase domain of ILK at atomic level. Structural changes with increasing concentrations of urea were analysed, and we observed a significant increase in the root mean square deviation, root mean square fluctuations, solvent accessible surface area and radius of gyration. A correlation was observed between in vitro and in silico studies.