Tumor Suppressor p16: Determination of Solution Structure and Analyses of Its Interaction with Cyclin-Dependent Kinase

to a variety of cellular proteins such as NF-␬B and I␬B (Hirai et al., 1994; Suzuki et al., 1996). Figure 1 shows the primary sequence of p16, along with that of other proteins in the p16 family: p15, p18, and p19, both human and murine. P16 is comprised mainly of four ankyrin repeats, a structural motif about Campus Chemical Instrument Center 33 amino acids long present in a variety of proteins in The Ohio State University a diverse range of organisms. This motif is believed to Columbus, Ohio 43210 be involved in protein–protein interactions (Lux et al. Summary As described previously (Tevelev et al., 1996), p16 displays a strong tendency to aggregate at high concen-The solution structure of the tumor suppressor p16 INK4A tration. This aggregation is often followed by denatur-has been determined by NMR, and important recogni-ation of the protein as observed by NMR. Furthermore, tion regions of both cdk4 and p16 INK4A have been identi-the structure of p16 appears to be very flexible as dem-fied. The tertiary structure of p16 INK4A contains four onstrated by rapid deuterium exchange of almost all helix-turn-helix motifs linked by three loops. Twelve protons during NMR studies in 2 H2O. These two inherent tumorigenic mutants of p16 INK4A have been constructed properties of p16 have made the structural determina-and analyzed for their structure and activity, and new tion by NMR (and possibly X-ray) extremely difficult. mutants have been designed rationally. A fragment of Using NMR samples at concentrations as low as 0.2 58 residues at the N terminus of cdk4 important for mM, we completed the total assignment and determined p16 INK4A binding has been identified. The importance the secondary structures of p16/⌬1–8 (a truncated form of this region was further verified by mutational analy-of p16, with the first 8 residues deleted). These results sis of cdk4. These results and docking experiments were the first structural analysis of an ankyrin repeat, have been used to assess possible modes of binding and demonstrated that the repeat exists in helix-turn-between p16 INK4A and cdk4. helix structures (Tevelev et al., 1996). Subsequently, the structures of two other ankyrin proteins have been re-Introduction ported: 53BP2 (Gorina and Pavletich, 1996) and p19 (Luh et al., 1997). Although p19 is a member of the p16 family, p16 and A negative regulator of the G 1-to-S transition, p16 INK4A p15 are the only proven tumor suppressors in this family (also …