Dynamic anticipation by Cdk2/Cyclin A-bound p27 mediates signal integration in cell cycle regulation.

p27Kip1 is an intrinsically disordered protein (IDP) that inhibits cyclin-dependent kinase (Cdk)/cyclin complexes (e.g., Cdk2/cyclin A), causing cell cycle arrest. Cell division progresses when stably Cdk2/cyclin A-bound p27 is phosphorylated on one or two structurally occluded tyrosine residues and a distal threonine residue (T187), triggering degradation of p27. Here, using an integrated biophysical approach, we show that Cdk2/cyclin A-bound p27 samples lowly-populated conformations that provide access to the non-receptor tyrosine kinases, BCR-ABL and Src, which phosphorylate Y88 or Y88 and Y74, respectively, thereby promoting intra-assembly phosphorylation (of p27) on distal T187. Even when tightly bound to Cdk2/cyclin A, intrinsic flexibility enables p27 to integrate and process signaling inputs, and generate outputs including altered Cdk2 activity, p27 stability, and, ultimately, cell cycle progression. Intrinsic dynamics within multi-component assemblies may be a general mechanism of signaling by regulatory IDPs, which can be subverted in human disease

Subdomain Architecture and Stability of a Giant Repeat Protein

Tandem repeat proteins, which are widespread in the human genome, tend to exhibit high stability and favorable expression, and hence, they are emerging as promising protein scaffolds in alternative to antibodies in biotechnology. In order to investigate the origin of the stability of these proteins, we dissect the subdomain architecture of the giant repeat protein PR65/A, which comprises 15 α-helical HEAT repeats, using a series of truncations and deletions. We find that the N (HEAT 1–2) and the C (HEAT 14–15) subdomains are not capable of independent folding, but the addition of HEAT 13 to HEAT 14–15 results in an independently stable C-terminal subdomain (HEAT 13–15), which is in turn further stabilized by the inclusion of HEAT 12 (HEAT 12–15). We also further show that the stability of HEAT 13–15 is enhanced by its fusion to HEAT 1–2, and the artificial 5-HEAT-repeat protein thereby created (HEAT NC) behaves like a cooperative multidomain protein. We construct further variants, lacking one or both of the terminal subdomains, and find that such subdomains function as stabilizing caps within full-length PR65/A as in their absence, the central subdomain of the protein unfolds to form non-native β-sheet-like oligomers. Taken together, our results suggest that in full-length PR65/A, the more unstable regions within the central repeats are protected by the adjacent folded repeats, which thus act as gatekeepers by virtue of their greater stability

Complex Energy Landscape of a Giant Repeat Protein

Here, we reveal a remarkable complexity in the unfolding of giant HEAT-repeat protein PR65/A, a molecular adaptor for the heterotrimeric PP2A phosphatases. The repeat array ruptures at multiple sites, leading to intermediate states with noncontiguous folded subdomains. There is a dominant sequence of unfolding, which reflects a nonuniform stability distribution across the repeat array and can be rationalized by theoretical models accounting for heterogeneous contact density in the folded structure. Unfolding of certain intermediates is, however, competitive, leading to parallel unfolding pathways. The low-stability, central repeats sample unfolded conformations under physiological conditions, suggesting how folding directs function: certain regions present rigid motifs for molecular recognition, whereas others have the flexibility with which to broaden the search area, as in the fly-casting mechanism. Partial unfolding of PR65/A also impacts catalysis by altering the proximity of bound catalytic subunit and substrate. Thus, the repeat array orchestrates the assembly and activity of PP2A.Fil: Tsytlonok, Maksym. University Of Cambridge; Estados Unidos. Hutchison/MRC Research Centre; Reino UnidoFil: Craig, Patricio Oliver. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; Argentina. University Of California At San Diego; Estados UnidosFil: Sivertsson, Elin. University Of Cambridge; Reino UnidoFil: Serquera, David. Hutchison/MRC Research Centre; Reino UnidoFil: Perrett, Sarah. Chinese Academy Of Sciences; República de ChinaFil: Best, Robert B.. University Of Cambridge; Reino UnidoFil: Wolynes, Peter G.. Rice University; Estados UnidosFil: Itzhaki, Laura S.. University Of Cambridge; Reino Unid

Fructose-1,6-bisphosphate couples glycolytic flux to activation of Ras

Yeast and cancer cells share the unusual characteristic of favoring fermentation of sugar over respiration. We now reveal an evolutionary conserved mechanism linking fermentation to activation of Ras, a major regulator of cell proliferation in yeast and mammalian cells, and prime proto-oncogene product. A yeast mutant (tps1∆) with overactive influx of glucose into glycolysis and hyperaccumulation of Fru1,6bisP, shows hyperactivation of Ras, which causes its glucose growth defect by triggering apoptosis. Fru1,6bisP is a potent activator of Ras in permeabilized yeast cells, likely acting through Cdc25. As in yeast, glucose triggers activation of Ras and its downstream targets MEK and ERK in mammalian cells. Biolayer interferometry measurements show that physiological concentrations of Fru1,6bisP stimulate dissociation of the pure Sos1/H-Ras complex. Thermal shift assay confirms direct binding to Sos1, the mammalian ortholog of Cdc25. Our results suggest that the Warburg effect creates a vicious cycle through Fru1,6bisP activation of Ras, by which enhanced fermentation stimulates oncogenic potency.Yeast and cancer cells both favor sugar fermentation in aerobic conditions. Here the authors describe a conserved mechanism from yeast to mammals where the glycolysis intermediate fructose-1,6-bisphosphate binds Cdc25/Sos1 and couples increased glycolytic flux to increased Ras proto-oncoprotein activity.status: publishe