Gankyrin, a newly defined oncoprotein also known as PSMD10 and P28, functions as a dual-negative regulator of the two most prominent tumor suppressor pathways: the CDK/pRb and MDM2/P53 pathways. Its aberrant expression has been prevalently found in human hepatocellular carcinomas (HCC) and esophagus squamous cell carcinomas (ESCC), which indicates gankyrin is a potential diagnostic and therapeutic target in cancers. Gankyrin is an ankyrin repeat (AR) protein which is composed of seven ankyrin repeats. Each repeat of gankyrin exhibits a canonical helix-turn-helix conformation and these seven repeats are stacked together near linearly to form a helix bundle, in which the neighboring ankyrin repeats are linked by loops of varied size which orientate perpendicularly to the axes of the helices of ankyrin repeats.
Our previous studies showed that while both specific CDK4 inhibitor p16INK4A (P16, exclusively consists of four AR repeats) 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. In this study 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.
The second part of the research on gankyrin involves an important structural characteristic of AR proteins: the presence of TPLH tetrapeptide or a variant at the beginning of canonical helix-turn-helix motif. Hydrogen bonding involving the Thr and His residues in the same and between adjacent tetrapeptide motifs presumably contributes to the formation of a hydrogen-bonding network and consequently the stability of the molecule. Thus, we investigated the structural role of this TPLH network in AR proteins by studying gankyrin, an oncogenic protein composed of seven ARs and six TPLH tetrapeptides, and p16INK4a, a tumor suppressor with four ARs and no TPLH tetrapeptides. Our results show that disrupting the TPLH network in the middle by removing Thr or both Thr and His from AR4 and AR5 of gankyrin significantly decreases its stability in both chemical- and heat-induced unfolding. On the other hand, introducing the TPLH network in p16INK4a in the middle (on AR3) increases its conformational stability. Our results suggest that the hydrogen bonding between neighboring TPLHs stabilize the structure of AR proteins when the TPLH motifs are in the middle of a long stretch of ankyrin repeats.