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

Abstract

Within the tumor suppressor protein INK4 (inhibitor of cyclin-dependent kinase 4) family, p15^INK4B is the smallest and the only one whose structure has not been determined previously, probably due to the protein's conformational flexibility and instability. In this work, multidimensional NMR studies were performed on this protein. The first tertiary structure was built by comparative modeling with p16^INK4A as the template, followed by restrained energy minimization with NMR constraints (NOE and H-bonds). For this purpose, the solution structure of p16^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 assignments. The nonhelical regions showed major improvement with root-mean-square deviation (RMSD) improved from 1.23 to 0.68 Å for backbone heavy atoms. The completion of p15^INK4B coupled with refinement of p16^INK4A made it 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^INK4A-CDK6 complex. This is an important step toward a comprehensive understanding of the precise functional roles of each INK4 member.