The how's and why's of protein folding intermediates.

The nature and role of intermediates have been the subject of much heated debate in the field of protein folding. Historically, intermediates were viewed as essential stepping-stones that guide a protein through the folding process to the native state. However, with the experimental identification of numerous small proteins that fold rapidly without intermediates, and the emergence from computational studies of new conceptual frameworks, came the thinking that intermediates can act as energy sinks, kinetic traps that result in less efficient folding. Whether 'good' or 'bad', it is without doubt that folding intermediates provide valuable information to protein chemists: at equilibrium they help to delineate the subdomain architecture of a protein and the hierarchy of subdomain stabilities; under kinetic conditions they provide experimentalists with additional snapshots of the folding reaction and, thereby, fundamental mechanistic details that are often lacking in the case of two-state folders. Intermediates give us valuable insights into the fluctuations from the native structure that may be important in regulating biological function. Lastly, intermediates are often the critical species in misfolding processes that lead to aggregation and disease. Here we review what we have learnt after almost half a century of protein-folding research, and we question two fundamental tests of our understanding: do we know enough about how proteins fold to design folding mechanisms de novo and can we exploit our knowledge to modulate protein-folding mechanisms in the cell for therapeutic benefit?

Using FlAsH to probe conformational changes in a large HEAT repeat protein.

We have investigated the use of FlAsH, a small fluorogenic molecule that binds to tetracysteine motifs, to probe folding of the 15-HEAT repeat protein PR65A. PR65A is one of a special class of modular non-globular proteins known as tandem repeat proteins, which are composed of small structural motifs that stack to form elongated, one-dimensional architectures. We were able to introduce linear and bipartite tetracysteine motifs at several sites along the α-helical HEAT array of PR65A without disrupting the structure or stability. When the linear tetracysteine motif CCPGCC was used, FlAsH fluorescence reported globally on the folding of the protein. When the tetracysteine motif was displayed in bipartite mode through the engineering of pairs of cysteines on adjacent HEAT repeats, FlAsH fluorescence became a reporter of local conformation and of oligomerisation. Thus, by designing FlAsH-binding sites at different locations along the repeat array one can interrogate specific properties of PR65A, paving the way for structure-function analysis of this protein both in vitro and in the cell.

Specific Conformational Dynamics and Expansion Underpin a Multi-Step Mechanism for Specific Binding of p27 with Cdk2/Cyclin A.

The protein p27, a prominent regulatory protein in eukaryotes and an intrinsically disordered protein (IDP), regulates cell division by causing cell cycle arrest when bound in ternary complex with cyclin-dependent kinase (Cdk2) and cyclins (e.g., Cdk2/Cyclin A). We present an integrative study of p27 and its binding to Cdk2/Cyclin A complex by performing single-molecule multiparameter fluorescence spectroscopy, stopped-flow experiments, and molecular dynamics simulations. Our results suggest that unbound p27 adopts a compact conformation and undergoes conformational dynamics across several orders of magnitude in time (nano-to milliseconds), reflecting a multi-step mechanism for binding Cdk2/Cyclin A. Mutagenesis studies reveal that the region D1 in p27 plays a significant role in mediating the association kinetics, undergoing conformational rearrangement upon initial binding. Additionally, FRET experiments indicate an expansion of p27 throughout binding. The detected local and long-range structural dynamics suggest that p27 exhibits a limited binding surface in the unbound form, and stochastic conformational changes in D1 facilitate initial binding to Cdk2/Cyclin A complex. Furthermore, the post-kinase inhibitory domain (post-KID) region of p27 exchanges between distinct conformational ensembles: an extended regime exhibiting worm-like chain behavior, and a compact ensemble, which may protect p27 against nonspecific interactions. In summary, the binding interaction involves three steps: (i) D1 initiates binding, (ii) p27 wraps around Cdk2/Cyclin A and D2 binds, and (iii) the fully-formed fuzzy ternary complex is formed concomitantly with an extension of the post-KID region. An understanding of how the IDP nature of p27 underpins its functional interactions with Cdk2/Cyclin A provides insight into the complex binding mechanisms of IDPs and their regulatory mechanisms.

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.

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.

Single-molecule FRET reveals hidden complexity in a protein energy landscape.

Here, using single-molecule FRET, we reveal previously hidden conformations of the ankyrin-repeat domain of AnkyrinR, a giant adaptor molecule that anchors integral membrane proteins to the spectrin-actin cytoskeleton through simultaneous binding of multiple partner proteins. We show that the ankyrin repeats switch between high-FRET and low-FRET states, controlled by an unstructured "safety pin" or "staple" from the adjacent domain of AnkyrinR. Opening of the safety pin leads to unravelling of the ankyrin repeat stack, a process that will dramatically affect the relative orientations of AnkyrinR binding partners and, hence, the anchoring of the spectrin-actin cytoskeleton to the membrane. Ankyrin repeats are one of the most ubiquitous molecular recognition platforms in nature, and it is therefore important to understand how their structures are adapted for function. Our results point to a striking mechanism by which the order-disorder transition and, thereby, the activity of repeat proteins can be regulated.

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 ß-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.

A telomerase-associated RecQ protein-like helicase resolves telomeric G-quadruplex structures during replication.

It is well established that G-quadruplex DNA structures form at ciliate telomeres and their formation throughout the cell-cycle by telomere-end-binding proteins (TEBPs) has been analyzed. During replication telomeric G-quadruplex structure has to be resolved to allow telomere replication by telomerase. It was shown that both phosphorylation of TEBPß and binding of telomerase are prerequisites for this process, but probably not sufficient to unfold G-quadruplex structure in timely manner to allow replication to proceed. Here we describe a RecQ-like helicase required for unfolding of G-quadruplex structures in vivo. This helicase is highly reminiscent of human RecQ protein-like 4 helicase as well as other RecQ-like helicase found in various eukaryotes and E. coli. In situ analyses combined with specific silencing of either the telomerase or the helicase by RNAi and co-immunoprecipitation experiments demonstrate that this helicase is associated with telomerase during replication and becomes recruited to telomeres by this enzyme. In vitro assays showed that a nuclear extract prepared from cells in S-phase containing both the telomerase as well as the helicase resolves telomeric G-quadruplex structure. This finding can be incorporated into a mechanistic model about the replication of telomeric G-quadruplex structures during the cell cycle.