Tumor suppressor INK4: Comparisons of conformational properties between p16(INK4A) and p18(INK4C)

The INK4 (inhibitor of cyclin-dependent kinase 4) family consists of four t umor-suppressor proteins: p15(INK4B) p16(INK4A), p18(INK4C), and p19(INK4D) While their sequences and structures are highly homologous, they show appr eciable differences in conformational flexibility, stability, and aggregati on tendency. Here, p16 and p18 were First compared directly by NMR for line broadening and disappearance, then investigated by three different approac hes in search of the causes of these differences. From denaturation experim ents it was found that both proteins are marginally stable with low denatur ation stability (1.94 and 2.98 kcal/mol, respectively). Heteronuclear H-1-N -15 nuclear Overhauser enhancement measurements revealed very limited confo rmational flexibility on the pico- to nanosecond time-scale for both p16 an d p18. H/H-2 exchange of amide protons monitored by NMR on three proteins ( p16, p18 as well as p15), however, revealed markedly different rates in the order p18 < p16 less than or equal to p15. A subset of very slowly exchang ing residues (about 19 in total) was identified in p18, including 16 residu es in the region of the fourth ankyrin repeat, probably as a result of a st abilizing effect by the extra ankyrin repeat. Thus, while INK4 proteins may have similar low thermodynamic stability as well as limited flexibility on the pico- to nanosecond time-scale, they display pronounced differences in the conformational flexibility on the time-scale of minutes to hours. Furt her analyses suggested that differences in H/H-2 exchange rates reflect dif ferences in the kinetic stability of the INK4, proteins, which in turn is r elated to differences in the aggregation tendency. (C) 1999 Academic Press.