Tumor suppressor INK4: Determination of the solution structure of p18(INK4C) and demonstration of the functional significance of loops in p18(INK4C) and p16(INK4A)

Since the structures of several ankyrin-repeat proteins including the INK4 (inhibitor of cyclin-dependent kinase 4) family have been reported recently , the detailed structures and the functional roles of the loops have drawn considerable interest. This paper addresses the potential importance of the loops of ankyrin-repeat proteins in three aspects. First, the solution str ucture of p18(INK4C) was determined by NMR, and the loop structures were an alyzed in detail. The loops adapt nascent antiparallel beta-sheet structure s, but the positions are slightly different from those in the crystal struc ture. A detailed comparison between the solution structures of p16 and p18 has also been presented. The determination of the p18 solution structure ma de such detailed comparisons possible for the first time. Second, the {H-1, N-15}HSQC NMR experiment was used to probe the interactions between p18(IN K4C) and other proteins. The results suggest that p18(INK4C) interacts very weakly with dna K and glutathione S-transferase via the loops. The third a spect employed site specific mutagenesis and functional assays. Three mutan ts of p18 and 11 mutants of p16 were constructed to test functional importa nce of loops and helices. The results suggest that loop 2 is likely to be p art of the recognition surface of p18(INK4C) Or p16(INK4A) for CDK4, and th ey provide quantitative functional contributions of specific residues. Over all, our results enhance understanding of the structural and functional rol es of the loops in INK4 tumor suppressors in particular and in ankyrin-repe at proteins in general.