Protein-protein interactions usually involve a large number of residues; thus it 
is difficult to elucidate functional and structural roles of specific residues 
located in the interface. This problem is particularly challenging for ankyrin 
repeat proteins (ARs), which consist of linear arrays of small repeating units 
and play critical roles in almost every life process via protein-protein 
interactions, because the residues involved are discontinuously dispersed in 
both the ARs and their partners. Our previous studies showed that while both 
specific CDK4 inhibitor p16INK4A (P16) and gankyrin bind to cyclin-dependent 
kinase 4 (CDK4) in similar fashion, only P16 inhibits the kinase activity of 
CDK4. While this could explain why P16 is a tumor suppressor and gankyrin is 
oncogenic, the structural basis of these contrasting properties was unknown. 
Here we show that a double mutant of gankyrin, L62H/I79D, inhibits the kinase 
activity of CDK4, similar to P16, and such CDK4-inhibtory activity is associated 
with the I79D but not L62H mutation. In addition, mutations at I79 and L62 bring 
about a moderate decrease in the stability of gankyrin. Further structural and 
biophysical analyses suggest that the substitution of Ile79 with Asp leads to 
local conformational changes in loops I-III of gankyrin. Taken together, our 
results allow the dissection of the "protein-protein binding" and "CDK4 
inhibition" functions of P16, show that the difference between tumor suppressing 
and oncogenic functions of P16 and gankyrin, respectively, mainly resides in a 
single residue, and provide structural insight to the contrasting biological 
functions of the two AR proteins.