TUMOR-SUPPRESSOR P16(INK4A) - STRUCTURAL CHARACTERIZATION OF WILD-TYPE AND MUTANT PROTEINS BY NMR AND CIRCULAR-DICHROISM

The tumor suppressor p16(INK4A) with eight N-terminal amino acids dele ted (p16/Delta 1-8) was expressed in Escherichia coli without any fusi on artifacts and purified, The integrity of p16/Delta 1-8 was confirme d by mass spectrometry, and its activity was demonstrated by in vitro cdk4 inhibition assay. Various physical methods were used to character ize the molecular and structural properties of p16/Delta 1-8. The prot ein was found to oligomerize in vitro, as demonstrated by gel electrop horesis, mass spectrometry, and NMR. Various approaches, including cha nges of concentration and pH, additions of salts, detergents, and vari ous organic solvents, and construction of a C-terminal deletion mutant and a cysteine mutant were used to try to reduce the extent of oligom erization. Only decreasing the protein concentration was found to redu ce oligomerization. The affinity between p16 molecules in vivo was dem onstrated by the yeast two-hybrid system. The protein was found to be very unstable on the basis of urea- and guanidinium chloride-induced d enaturation studies monitored by NMR and CD, respectively. Despite the se unfavorable properties, total NMR assignments were accomplished wit h uniform C-13 and N-15 isotope labeling. All multidimensional NMR exp eriments were performed at a very low concentration of 0.2 mM. The sec ondary structure was then determined from the NMR data. The results of NMR and CD studies indicate that the protein is highly alpha-helical, and the ankyrin repeat sequences show helix-turn-helix structures. Th is is the first structural information obtained for the important moti f of ankyrin repeats, Overall, p16/Delta 1-8 appears to be conformatio nally flexible. In order to understand the structural basis of the fun ctional changes for some mutants existing in tumor cells, several miss ense mutants of p16/Delta 1-8 were constructed. Four of them were expr essed at high levels and purified, The molecular and structural proper ties of these mutants were analyzed by CD and NMR and compared with th e corresponding properties of wild-type p16/Delta 1-8. The results sug gest that the functional changes in P114L and G101W are likely to be r elated to global conformational changes. In addition, we have demonstr ated that the tendency of aggregation increases significantly by a sin gle D84H mutation.