Structural characterization of the tumor suppressor p16, an ankyrin‐like repeat protein

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 of human 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 pM. The CD spectrum indicates that the protein is composed of 33% a-helix, 22% P-sheet, 19% p-turn, and 27% other (which includes aromatic and random coil contributions). Further CD experiments suggest that p16 exhibits low structural stability with a AG of -2.3 kcal/mol. This weak stability is a consequence of a highly dynamic structure as measured by ANS-binding, NMR hydrogendeuterium exchange, and fluorescence. It is possible that a well-defined tertiary structure is imparted upon the binding of p16 to CDK4.Keywords: analytical ultracentrifugation; ankyrin-like repeat; cyclin; circular dichroism; cyclin dependent kinase inhibitor Control of the eukaryotic cell cycle is a highly regulated process that involves the interaction of a large number of macromolecules. Critical checkpoints in this process are found during the entry of cells into both the GUS and G2/M phases of the cell cycle (for reviews, see Draetta, 1994;Sherr, 1995). The cyclin-dependent kinases (CDKs) are enzymes whose activities govern these checkpoints. CDK holoenzymes, which consist of a CDK catalytic subunit and a cyclin regulatory subunit, are regulated, in part, by their temporal expression, degradation, phosphorylation, and dephosphorylation. This regulation throughout the cell cycle serves to drive the cell through the successive phases.Recent studies have identified CDK inhibitors, a new class of proteins, adding yet another level of control to CDK regulation (for review, see Elledge & Harper, 1994). These CDK inhibitors are thought to function by blocking and/or disrupting CDK-cyclin association. p16, one of the first of these inhibitors to be discovered, was originally cloned from a yeast-two hybrid interaction screen using CDK4 as the bait protein (Serrano et al., 1993).

Studies of the Ankyrin Repeats of the Drosophila melanogaster Notch Receptor. 1. Solution Conformational and Hydrodynamic Properties

“…The studies here examine participation in the ankyrin domain of repeats on the C-terminus, but not the N-terminus. Although the ellipticity of the Notch ankyrin polypeptides is lower than expected based on crystal structures of other ankyrin repeat proteins, the CD spectra are consistent both in shape and in magnitude with CD spectra of other ankyrin repeat domains (44,(72)(73)(74)(75), including p16, where high-resolution structural data indicate that each ankyrin repeat adopts an ordered ankyrin fold (41,76).…”

Repeat-protein folding: New insights into origins of cooperativity, stability, and topology

“…Tsai and coworkers found this protein to display a steep, cooperative unfolding transition when helix content was monitored by circular dichroism (CD) spectroscopy as a function of guanidine hydrochloride, although the midpoint for the unfolding transition was rather low [46]. Similar results were obtained using urea [47] and thermal denaturation [48]. Later, Itzhaki's group examined the unfolding transition of this protein more extensively [49], and showed that similar unfolding transitions (and fitted thermodynamic parameters) are obtained when urea-induced unfolding is monitored by CD, fluorescence from two N-and C-terminal tryptophans, and gel filtration chromatography The Drosophila Notch ankyrin domain also appears to unfold via a cooperative, all-or-none mechanism.…”

Experimental Characterization of the Folding Kinetics of the Notch Ankyrin Domain

“…Despite their modular architecture, several linear repeat proteins have been shown to display two-state equilibrium unfolding, including the four-ankyrin-repeat proteins p16 INK4a and myotrophin, [30][31][32] and the seven-ankyrin-repeat domain from the Drosophila Notch receptor. 33,34 Recently, a three-repeat designed ankyrin domain has been shown to conform to a simple kinetic two-state mechanism.…”