Tumor suppressor INK4: Refinement of p16(INK4A) structure and determination of p15(INK4B) structure by comparative modeling and NMR data

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kin ase 4) family, p15(INK4B) is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformat ional flexibility and instability. In this work, multidimensional NMR studi es were performed on this protein. The first tertiary structure was built b y comparative modeling with p16(INK4A) as the template, followed by restrai ned energy minimization with NMR constraints (NOE and H-bonds). For this pu rpose, the solution structure of F16(INK4A), whose quality was also limited by similar problems, was refined with additional NMR experiments conducted on an 800 MHz spectrometer and by structure-based iterative NOE assignment s. The nonhelical regions showed major improvement with root-mean square de viation (RMSD) improved from 1.23 to 0.68 Angstrom for backbone heavy atoms . The completion of p15(INK4B) coupled with refinement of p16(INK4A) made i t possible to compare the structures of the four INK4 members in depth. and to compare the structures of p16(INK4A) in the free form and in the p16(IN K4A)-CDK6 complex. This is an important step toward a comprehensive underst anding of the precise functional roles of each INK4 member.