Intrinsically disordered protein biosensor tracks the physical-chemical effects of osmotic stress on cells.

Cell homeostasis is perturbed when dramatic shifts in the external environment cause the physical-chemical properties inside the cell to change. Experimental approaches for dynamically monitoring these intracellular effects are currently lacking. Here, we leverage the environmental sensitivity and structural plasticity of intrinsically disordered protein regions (IDRs) to develop a FRET biosensor capable of monitoring rapid intracellular changes caused by osmotic stress. The biosensor, named SED1, utilizes the Arabidopsis intrinsically disordered AtLEA4-5 protein expressed in plants under water deficit. Computational modeling and in vitro studies reveal that SED1 is highly sensitive to macromolecular crowding. SED1 exhibits large and near-linear osmolarity-dependent changes in FRET inside living bacteria, yeast, plant, and human cells, demonstrating the broad utility of this tool for studying water-associated stress. This study demonstrates the remarkable ability of IDRs to sense the cellular environment across the tree of life and provides a blueprint for their use as environmentally-responsive molecular tools

Mechanism of the Interaction between the Intrinsically Disordered C-Terminus of the Pro-Apoptotic ARTS Protein and the Bir3 Domain of XIAP

ARTS (Sept4_i2) is a mitochondrial pro-apoptotic protein that functions as a tumor suppressor. Its expression is significantly reduced in leukemia and lymphoma patients. ARTS binds and inhibits XIAP (X-linked Inhibitor of Apoptosis protein) by interacting with its Bir3 domain. ARTS promotes degradation of XIAP through the proteasome pathway. By doing so, ARTS removes XIAP inhibition of caspases and enables apoptosis to proceed. ARTS contains 27 unique residues in its C-terminal domain (CTD, residues 248–274) which are important for XIAP binding. Here we characterized the molecular details of this interaction. Biophysical and computational methods were used to show that the ARTS CTD is intrinsically disordered under physiological conditions. Direct binding of ARTS CTD to Bir3 was demonstrated using NMR and fluorescence spectroscopy. The Bir3 interacting region in ARTS CTD was mapped to ARTS residues 266–274, which are the nine C-terminal residues in the protein. Alanine scan of ARTS 266–274 showed the importance of several residues for Bir3 binding, with His268 and Cys273 contributing the most. Adding a reducing agent prevented binding to Bir3. A dimer of ARTS 266–274 formed by oxidation of the Cys residues into a disulfide bond bound with similar affinity and was probably required for the interaction with Bir3. The detailed analysis of the ARTS – Bir3 interaction provides the basis for setting it as a target for anti cancer drug design: It will enable the development of compounds that mimic ARTS CTD, remove IAPs inhibition of caspases, and thereby induce apoptosis

Specific Recognition of p53 Tetramers by Peptides Derived from p53 Interacting Proteins

Oligomerization plays a major role in regulating the activity of many proteins, and in modulating their interactions. p53 is a homotetrameric transcription factor that has a pivotal role in tumor suppression. Its tetramerization domain is contained within its C-terminal domain, which is a site for numerous protein-protein interactions. Those can either depend on or regulate p53 oligomerization. Here we screened an array of peptides derived from proteins known to bind the tetrameric p53 C-terminal domain (p53CTD) and identified ten binding peptides. We quantitatively characterized their binding to p53CTD using fluorescence anisotropy. The peptides bound tetrameric p53CTD with micromolar affinities. Despite the high charge of the binding peptides, electrostatics contributed only mildly to the interactions. NMR studies indicated that the peptides bound p53CTD at defined sites. The most significant chemical shift deviations were observed for the peptides WS100B(81–92), which bound directly to the p53 tetramerization domain, and PKCα(281–295), which stabilized p53CTD in circular dichroism thermal denaturation studies. Using analytical ultracentrifugation, we found that several of the peptides bound preferentially to p53 tetramers. Our results indicate that the protein-protein interactions of p53 are dependent on the oligomerization state of p53. We conclude that peptides may be used to regulate the oligomerization of p53

Crowding Alone Cannot Account for Cosolute Effect on Amyloid Aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed “macromolecular crowding”. Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation

Structural Preferences Shape the Entropic Force of Disordered Protein Ensembles.

Intrinsically disordered protein regions (IDRs) make up over 30% of the human proteome and exist in a dynamic conformational ensemble instead of a native, well-folded structure. Tethering IDRs to a surface (for example, the surface of a well-folded region of the same protein) can reduce the number of accessible conformations in these ensembles. This reduces the ensembles conformational entropy, generating an effective entropic force that pulls away from the point of tethering. Recent experimental work has shown that this entropic force causes measurable, physiologically relevant changes to protein function. But how the magnitude of this force depends on IDR sequence remains unexplored. Here, we use all-atom simulations to analyze how structural preferences in IDR ensembles contribute to the entropic force they exert upon tethering. We show that sequence-encoded structural preferences play an important role in determining the magnitude of this force: compact, spherical ensembles generate an entropic force that can be several times higher than more extended ensembles. We further show that changes in the surrounding solutions chemistry can modulate the IDR entropic force strength. We propose that the entropic force is a sequence-dependent, environmentally tunable property of terminal IDR sequences