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Periodic Report Summary 1 - STARIDP (Structure and function of intrinsically disordered proteins (IDPs) in cell cycle regulation)

The EU funded project STARIDP focused on the understanding of the cell-cycle regulation by an intrinsically disordered inhibitor protein (IDP) p27.
The intrinsic flexibility of p27 and other IDPs affords functional advantages in molecular recognition. p27 is able for example to regulate eukaryotic cell division by interacting with a number of cyclin-dependent kinase (Cdk)/cyclin complexes, as well as with other nuclear and cytoplasmic targets. Phosphorylation of p27 by oncogenic kinases contributes to tumorigenesis in several human cancers. The crystal structure of p27 bound to Cdk/Cyclin complex and ensemble measurements provide molecular details of the specificity and the sequential induced-folding mechanism of this interaction. However, the mechanistic details of the dynamic processes associated with the phosphorylation of p27 required for the degradation of p27 are not completely understood. Our aim is to characterize in more detail the mechanistic process of p27 interaction with Cdk2/CyclinA complex using stopped-flow kinetic experiments and single-molecule Förster resonance energy transfer (smFRET).
In the work carried out so far in this project, we have extensively analysed the mechanistic process of p27 interaction with Cdk2/CyclinA complex using stopped-flow kinetic experiments and single-molecule Förster resonance energy transfer (smFRET) studies. Our results suggest that some regions in p27 become stretched upon interaction rather than more common assumption of compaction upon binding. Moreover, we find that fully disordered region D1 in p27KID serves as a recognition domain for the previously not characterized initial weak interaction with CyclinA. This binding occurs via a conformational selection mechanism and guides the interaction pathway towards a very tight inhibitory complex via an additional induced-fit mechanism.

First of all, we addressed the question on how the conformation of p27 changes upon interacting with Cdk2/CyclinA complex we performed smFRET measurements on the free and bound p27. We introduced three pairs of donor-acceptor combinations at positions C29-C54, C54-C93 and C75-C110 to follow the distance-related changes between the dye-pair upon binding. The data show that the distance between the positions covered in p27 increases significantly upon binding to Cdk2/CyclinA complex. These data suggest that p27 is experiencing a global unfolding or stretching upon interaction while some elements undergo disorder-to-order transition at a level of secondary structure.

Secondly, we tried to understand the mechanistic details of the transition between unfolded and bound but stretched conformation of p27KID, we performed a serious of rapid-mixing experiments. Intrinsic tryptophan fluorescence in CyclinA, Cdk2 and p27KID allowed direct monitoring of inhibitor kinetics. In the binding kinetics of p27KID with CyclinA we detected a very fast and a slow phase associated with fluorescence decrease and fluorescence increase, respectively, which point to at least a three-step mechanism involving an intermediate in accordance with previously published results.
The basal function of p27 is the inhibition of the kinase activity of Cdk/cyclin complexes, which can be relieved through phosphorylation of its tyrosine residue(s) by non-receptor tyrosine kinases (NRTKs). Relief of Cdk inhibition triggers several additional posttranslational modifications, primarily phosphorylation of a residue T187 within the same p27 molecule, which leads to its degradation, complete activation of Cdk/cyclin complexes, and transition to S phase in cell division.

Therefore, we studied the effect of sequential phosphorylation of p27 on its interaction with Cdk2/CyclinA complex using biochemical and single-molecule FRET and anisotropy methods. Our results provide some key answers to mechanistic question regarding the roles of IDPs as regulatory switches in signaling pathways; specifically, we addressed how sites within a bound IDP (p27 in complex with Cdk2/cyclin A) can become accessible to, and integrate, regulatory modification despite apparent steric inaccessibility within a rigid 3D structure. p27 experiences phosphorylation on two structurally inaccessible tyrosines, which signal p27 degradation and promote cell division. Here we have shown that the two events are sequential: the phosphorylation of Y88 is required for the subsequent phosphorylation of Y74, resulting in the D2 region of p27 to be fully displaced from Cdk2.

Using single-molecule fluorescence methods, we show that the priming phosphorylation of Y88 is only possible because this region structurally fluctuates between a major bound and a minor partially released state compatible with NRTK-mediated phosphorylation. Upon phosphorylation, the solvent-exposed conformation for Y88 becomes the major state, and sterically enables phosphorylation of Y74, which displaces the D2 subdomain of p27 and facilitates intra-complex phosphorylation of another residue, T187, which mediates p27 poly-ubiquitination and degradation and, ultimately, cell cycle progression. In all, tightly bound p27 within its complex with Cdk2/cyclin A dynamically anticipates the conformational changes caused by sequential phosphorylation. These structural fluctuations and remodeling of the conformational ensemble enable Cdk2/cyclin A-bound p27 to actively integrate upstream kinase signals with rheostat-like precision.

In all, our data are consistent with a model of dynamic anticipation and sequential remodeling of the conformational energy landscape of bound p27, which might represent a general mechanistic theme of how IDPs sense, integrate and propagate signals within signaling pathways. Our results also show that conventional stopped-flow fluorescence in combination with novel single-molecule fluorescence methods can provide a detailed mechanism of action of IDPs in the cells. This will serve as an example on how the mechanisms of oncogenic proteins (either ordered or disordered) can be analyzed in detail for identification of ways for intervention with medicinal potential.

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