Explore the vast range of titles available.

Phosphorylation of intrinsically disordered proteins (IDPs) can produce changes in structural and dynamical properties and thereby mediate critical biological functions. How phosphorylation effects intrinsically disordered proteins has been studied for an increasing number of IDPs, but a systematic understanding is still lacking. Here, we compare the collapse propensity of four disordered proteins, Ash1, the C-terminal domain of RNA polymerase (CTD2’), the cytosolic domain of E-Cadherin, and a fragment of the p130Cas, in unphosphorylated and phosphorylated forms using extensive all-atom molecular dynamics (MD) simulations. We find all proteins to show V-shape changes in their collapse propensity upon multi-site phosphorylation according to their initial net charge: phosphorylation expands neutral or overall negatively charged IDPs and shrinks positively charged IDPs. However, force fields including those tailored towards and commonly used for IDPs overestimate these changes. We find quantitative agreement of MD results with SAXS and NMR data for Ash1 and CTD2’ only when attenuating protein electrostatic interactions by using a higher salt concentration ( e . g . 350 mM), highlighting the overstabilization of salt bridges in current force fields. We show that phosphorylation of IDPs also has a strong impact on the solvation of the protein, a factor that in addition to the actual collapse or expansion of the IDP should be considered when analyzing SAXS data. Compared to the overall mild change in global IDP dimension, the exposure of active sites can change significantly upon phosphorylation, underlining the large susceptibility of IDP ensembles to regulation through post-translational modifications.

Ferroptosis is a type of cell death caused by radical-driven lipid peroxidation, leading to membrane damage and rupture. Here we show that enzymatically produced sulfane sulfur (S 0 ) species, specifically hydropersulfides, scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis. By providing sulfur for S 0 biosynthesis, cysteine can support ferroptosis resistance independently of the canonical GPX4 pathway. Our results further suggest that hydropersulfides terminate radical chain reactions through the formation and self-recombination of perthiyl radicals. The autocatalytic regeneration of hydropersulfides may explain why low micromolar concentrations of persulfides suffice to produce potent cytoprotective effects on a background of millimolar concentrations of glutathione. We propose that increased S 0 biosynthesis is an adaptive cellular response to radical-driven lipid peroxidation, potentially representing a primordial radical protection system. Enzymatically generated sulfane sulfur species called hydropersulfides terminate free radical chain reactions to prevent oxidative membrane damage and ferroptosis induction.

c-Abl kinase, a key signaling hub in many biological processes ranging from cell development to proliferation, is tightly regulated by two inhibitory Src homology domains. An N-terminal myristoyl modification can bind to a hydrophobic pocket in the kinase C-lobe, which stabilizes the autoinhibitory assembly. Activation is triggered by myristoyl release. We used molecular dynamics simulations to show how both myristoyl and the Src homology domains are required to impose the full inhibitory effect on the kinase domain and reveal the allosteric transmission pathway at residue-level resolution. Importantly, we find myristoyl insertion into a membrane to thermodynamically compete with binding to c-Abl. Myristoyl thus not only localizes the protein to the cellular membrane, but membrane attachment at the same time enhances activation of c-Abl by stabilizing its preactivated state. Our data put forward a model in which lipidation tightly couples kinase localization and regulation, a scheme that currently appears to be unique for this non-receptor tyrosine kinase.

Mechanosensing at focal adhesions regulates vital cellular processes. Here, we present results from molecular dynamics (MD) and mechano-biochemical network simulations that suggest a direct role of Focal Adhesion Kinase (FAK) as a mechano-sensor. Tensile forces, propagating from the membrane through the PIP2 binding site of the FERM domain and from the cytoskeleton-anchored FAT domain, activate FAK by unlocking its central phosphorylation site (Tyr576/577) from the autoinhibitory FERM domain. Varying loading rates, pulling directions, and membrane PIP2 concentrations corroborate the specific opening of the FERM-kinase domain interface, due to its remarkably lower mechanical stability compared to the individual alpha-helical domains and the PIP2-FERM link. Analyzing downstream signaling networks provides further evidence for an intrinsic mechano-signaling role of FAK in broadcasting force signals through Ras to the nucleus. This distinguishes FAK from hitherto identified focal adhesion mechano-responsive molecules, allowing a new interpretation of cell stretching experiments.

As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes. The existence, nature and biological relevance of mechanoradicals in proteins are unknown. Here authors show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species and suggest that collagen I evolved as a radical sponge against mechano-oxidative damage.

Unfolded states of proteins and native states of intrinsically disordered proteins (IDPs) populate heterogeneous conformational ensembles in solution. The average sizes of these heterogeneous systems, quantified by the radius of gyration (RG ), can be measured by small-angle X-ray scattering (SAXS). Another parameter, the mean dye-to-dye distance (RE ) for proteins with fluorescently labeled termini, can be estimated using single-molecule Förster resonance energy transfer (smFRET). A number of studies have reported inconsistencies in inferences drawn from the two sets of measurements for the dimensions of unfolded proteins and IDPs in the absence of chemical denaturants. These differences are typically attributed to the influence of fluorescent labels used in smFRET and to the impact of high concentrations and averaging features of SAXS. By measuring the dimensions of a collection of labeled and unlabeled polypeptides using smFRET and SAXS, we directly assessed the contributions of dyes to the experimental values RG and RE . For chemically denatured proteins we obtain mutual consistency in our inferences based on RG and RE , whereas for IDPs under native conditions, we find substantial deviations. Using computations, we show that discrepant inferences are neither due to methodological shortcomings of specific measurements nor due to artifacts of dyes. Instead, our analysis suggests that chemical heterogeneity in heteropolymeric systems leads to a decoupling between RE and RG that is amplified in the absence of denaturants. Therefore, joint assessments of RG and RE combined with measurements of polymer shapes should provide a consistent and complete picture of the underlying ensembles.

The talin-vinculin axis is a key mechanosensing component of cellular focal adhesions. How talin and vinculin respond to forces and regulate one another remains unclear. By combining single-molecule magnetic tweezers experiments, Molecular Dynamics simulations, actin-bundling assays, and adhesion assembly experiments in live cells, we here describe a two-ways allosteric network within vinculin as a regulator of the talin-vinculin interaction. We directly observe a maturation process of vinculin upon talin binding, which reinforces the binding to talin at a rate of 0.03 s −1 . This allosteric transition can compete with force-induced dissociation of vinculin from talin only at forces up to 10 pN. Mimicking the allosteric activation by mutation yields a vinculin molecule that bundles actin and localizes to focal adhesions in a force-independent manner. Hence, the allosteric switch confines talin-vinculin interactions and focal adhesion build-up to intermediate force levels. The ‘allosteric vinculin mutant’ is a valuable molecular tool to further dissect the mechanical and biochemical signalling circuits at focal adhesions and elsewhere. Vinculin binding to talin is a key event in focal adhesion dynamics; yet, how vinculin is activated to recruit actin remains unknown. Here, the authors use a multiscale approach to reveal that talin activates vinculin through an intricate allosteric mechanism tightly regulated by force.

Collagen is a force-bearing, hierarchical structural protein important to all connective tissue. In tendon collagen, high load even below macroscopic failure level creates mechanoradicals by homolytic bond scission, similar to polymers. The location and type of initial rupture sites critically decide on both the mechanical and chemical impact of these micro-ruptures on the tissue, but are yet to be explored. We here use scale-bridging simulations supported by gel electrophoresis and mass spectrometry to determine breakage points in collagen. We find collagen crosslinks, as opposed to the backbone, to harbor the weakest bonds, with one particular bond in trivalent crosslinks as the most dominant rupture site. We identify this bond as sacrificial, rupturing prior to other bonds while maintaining the material’s integrity. Also, collagen’s weak bonds funnel ruptures such that the potentially harmful mechanoradicals are readily stabilized. Our results suggest this unique failure mode of collagen to be tailored towards combatting an early onset of macroscopic failure and material ageing. Collagen is an important structural protein in connective tissue, but the effect of location and type of micro-ruptures in the structure on the overall tissue is not well understood. Here, the authors use scale-bridging simulations to determine the breakage points in collagen, and how the failure mode helps to prevent material ageing

Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole. The mechanisms through which SAS-6 polymerization ensures centriole organelle architecture remain elusive. We deploy photothermally-actuated off-resonance tapping high-speed atomic force microscopy to decipher surface SAS-6 self-assembly mechanisms. We show that the surface shifts the reaction equilibrium by ~10 4 compared to solution. Moreover, coarse-grained molecular dynamics and atomic force microscopy reveal that the surface converts the inherent helical propensity of SAS-6 polymers into 9-fold rings with residual asymmetry, which may guide ring stacking and impart chiral features to centrioles and cilia. Overall, our work reveals fundamental design principles governing centriole assembly. The centriole exhibits an evolutionarily conserved 9-fold radial symmetry that stems from a cartwheel containing vertically stacked ring polymers that harbor 9 homodimers of the protein SAS-6. Here the authors show how dual properties inherent to surface-guided SAS-6 self-assembly possess spatial information that dictates correct scaffolding of centriole architecture.

How proteins harness mechanical force to control function is a significant biological question. Here we describe a human cell surface receptor that couples ligand binding and force to trigger a chemical event which controls the adhesive properties of the receptor. Our studies of the secreted platelet oxidoreductase, ERp5, have revealed that it mediates release of fibrinogen from activated platelet αIIbβ3 integrin. Protein chemical studies show that ligand binding to extended αIIbβ3 integrin renders the βI-domain Cys177-Cys184 disulfide bond cleavable by ERp5. Fluid shear and force spectroscopy assays indicate that disulfide cleavage is enhanced by mechanical force. Cell adhesion assays and molecular dynamics simulations demonstrate that cleavage of the disulfide induces long-range allosteric effects within the βI-domain, mainly affecting the metal-binding sites, that results in release of fibrinogen. This coupling of ligand binding, force and redox events to control cell adhesion may be employed to regulate other protein-protein interactions. Many proteins embedded in a cell’s surface allow the cell to interact with its surroundings. Integrins are a group of cell surface proteins that have many uses in different cells. Integrins become activated when they come into contact with other specific proteins, which like other molecules that bind to proteins are referred to collectively as “ligands”. Much research has focused on how ligands become attached to integrins and how this activates these cell surface proteins. Yet how integrins release ligands and become inactive has not been studied before. One type of integrin, called αIIbβ3, is involved in blood clotting. Found on the surface of blood platelets – the fragments of cells in the blood that play a central role in clotting, this integrin binds to a ligand called fibrinogen. Fibrinogen links platelets together to form clots by building bridges between integrins. Passam, Chiu et al. have now studied platelets from donated human blood to understand how the integrin αIIbβ3 disengages from fibrinogen. The investigation showed that an enzyme called ERp5 aids the release of fibrinogen from the integrin. ERp5 can be released by blood vessel walls and by activated platelets. The experiments revealed that ERp5 breaks a chemical link, called a disulfide bond, in the integrin, but only when the protein is already bound to its ligand. Breaking the disulfide bond (a chemical process known as reduction) changes the integrin’s structure so that it lets go of fibrinogen. Moreover, when physical forces such as blood flow put the integrin under strain, the ERp5 enzyme becomes more effective. These findings show how ligand binding and mechanical force work together to control the breaking of a chemical bond in a human integrin. This chemical event then in turn controls the release of the integrin's ligand. It is possible that other protein-protein interactions may involve similar mechanisms, but this remains to be explored. Lastly, Passam, Chiu et al. suggest that the release of fibrinogen might help to limit the growth of blood clots so they do not block the blood vessels. Further studies should test this hypothesis. Inappropriate clotting can have severe health effects including heart attacks and strokes. As such, this investigation may hint at a more subtle way to regulate clotting through integrin αIIbβ3, such as boosting fibrinogen release to see if it helps slow or reduce clotting without stopping it altogether.