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Structural characterization of the tumor suppressor p16, an ankyrin-like repeat protein
1Division of Macromolecular Structure, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000, USA.
Abstract:
The p16 protein has been identified as a tumor suppressor that functions by inhibiting the cyclin-dependent kinases CDK4 and CDK6. Deletions or point mutations in the p16 gene have been found in a number cancers, emphasizing its importance in regulating cell cycle progression. Inhibition by p16 occurs through protein-protein interactions with its targets. This is not surprising, since p16 is thought to contain ankyrin-like repeats, motifs implicated in protein-protein interactions. Our goal was to identify structural characteristics of p16 not only as an important step towards understanding CDK4 inhibition but also to explore the role of ankyrin repeats in the p16 structure, as no detailed structure of any protein containing these motifs has been reported. We have expressed, refolded, and purified p16 from E. coli and have shown it to be functionally active by specific binding to CDK4. Analytical ultracentrifugation has shown that p16 weakly self-associates to form dimers with a Kd = 270 microM. The CD spectrum indicates that the protein is composed of 33% alpha-helix, 22% beta-sheet, 19% beta-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a delta G of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogen-deuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.
Insights
The p16 protein, a tumor suppressor, was structurally characterized to understand its cell cycle inhibition mechanism. Researchers found p16 is dynamic and weakly stable, suggesting binding to CDK4 may stabilize its structure.
Area of Science:
- Molecular Biology
- Structural Biology
- Cancer Research
Background:
- The p16 protein acts as a tumor suppressor by inhibiting cyclin-dependent kinases CDK4 and CDK6, crucial for cell cycle progression.
- Mutations in the p16 gene are linked to various cancers, highlighting its critical role in cell cycle regulation.
- p16's function involves protein-protein interactions, potentially mediated by ankyrin-like repeats, but its detailed structure was unknown.
Purpose of the Study:
- To elucidate the structural characteristics of the p16 protein.
- To understand the mechanism of CDK4 inhibition by p16.
- To explore the role of ankyrin repeats in p16 structure and function.
Main Methods:
- p16 protein expression, refolding, and purification from E. coli.
- Functional assessment via specific binding assays with CDK4.
- Structural analysis using analytical ultracentrifugation, circular dichroism (CD) spectroscopy, ANS-binding, NMR hydrogen-deuterium exchange, and fluorescence.
Main Results:
- Purified p16 protein demonstrated functional activity through specific binding to CDK4.
- Analytical ultracentrifugation revealed weak p16 self-association into dimers (Kd = 270 microM).
- CD spectroscopy indicated a composition of 33% alpha-helix, 22% beta-sheet, 19% beta-turn, and 27% other; p16 exhibited low structural stability (ΔG = -2.3 kcal/mol) and high dynamics.
Conclusions:
- The p16 protein possesses a dynamic and weakly stable structure.
- The findings suggest that p16's tertiary structure may become well-defined upon binding to CDK4.
- This structural insight is crucial for understanding p16's role in cell cycle regulation and cancer.