How TriC folds tricky proteins.

How chaperonins orchestrate the successful folding of even the most elaborate of proteins is a question of central importance. In two recent studies in Cell by Joachimiak et al. and Freund et al., a new class of TRiC substrate is identified, and how the chaperonin exploits its different subunits to extend its substrate repertoire and direct productive folding is revealed.

An interdomain energetic tug-of-war creates the allosterically active state in Hsp70 molecular chaperones.

The allosteric mechanism of Hsp70 molecular chaperones enables ATP binding to the N-terminal nucleotide-binding domain (NBD) to alter substrate affinity to the C-terminal substrate-binding domain (SBD) and substrate binding to enhance ATP hydrolysis. Cycling between ATP-bound and ADP/substrate-bound states requires Hsp70s to visit a state with high ATPase activity and fast on/off kinetics of substrate binding. We have trapped this "allosterically active" state for the E. coli Hsp70, DnaK, and identified how interactions among the NBD, the ß subdomain of the SBD, the SBD α-helical lid, and the conserved hydrophobic interdomain linker enable allosteric signal transmission between ligand-binding sites. Allostery in Hsp70s results from an energetic tug-of-war between domain conformations and formation of two orthogonal interfaces: between the NBD and SBD, and between the helical lid and the ß subdomain of the SBD. The resulting energetic tension underlies Hsp70 functional properties and enables them to be modulated by ligands and cochaperones and "tuned" through evolution.

Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands.

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding.

Substrate-binding domain conformational dynamics mediate Hsp70 allostery.

Binding of ATP to the N-terminal nucleotide-binding domain (NBD) of heat shock protein 70 (Hsp70) molecular chaperones reduces the affinity of their C-terminal substrate-binding domain (SBD) for unfolded protein substrates. ATP binding to the NBD leads to docking between NBD and ßSBD and releasing of the α-helical lid that covers the substrate-binding cleft in the SBD. However, these structural changes alone do not fully account for the allosteric mechanism of modulation of substrate affinity and binding kinetics. Through a multipronged study of the Escherichia coli Hsp70 DnaK, we found that changes in conformational dynamics within the ßSBD play a central role in interdomain allosteric communication in the Hsp70 DnaK. ATP-mediated NBD conformational changes favor formation of NBD contacts with lynchpin sites on the ßSBD and force disengagement of SBD strand ß8 from strand ß7, which leads to repacking of a ßSBD hydrophobic cluster and disruption of the hydrophobic arch over the substrate-binding cleft. In turn, these structural rearrangements drastically enhance conformational dynamics throughout the entire ßSBD and particularly around the substrate-binding site. This negative, entropically driven allostery between two functional sites of the ßSBD-the NBD binding interface and the substrate-binding site-confers upon the SBD the plasticity needed to bind to a wide range of chaperone clients without compromising precise control of thermodynamics and kinetics of chaperone-client interactions.

Allosteric signal transmission in the nucleotide-binding domain of 70-kDa heat shock protein (Hsp70) molecular chaperones.

The 70-kDa heat shock protein (Hsp70) chaperones perform a wide array of cellular functions that all derive from the ability of their N-terminal nucleotide-binding domains (NBDs) to allosterically regulate the substrate affinity of their C-terminal substrate-binding domains in a nucleotide-dependent mechanism. To explore the structural origins of Hsp70 allostery, we performed NMR analysis on the NBD of DnaK, the Escherichia coli Hsp70, in six different states (ligand-bound or apo) and in two constructs, one that retains the conserved and functionally crucial portion of the interdomain linker (residues ) and another that lacks the linker. Chemical-shift perturbation patterns identify residues at subdomain interfaces that constitute allosteric networks and enable the NBD to act as a nucleotide-modulated switch. Nucleotide binding results in changes in subdomain orientations and long-range perturbations along subdomain interfaces. In particular, our findings provide structural details for a key mechanism of Hsp70 allostery, by which information is conveyed from the nucleotide-binding site to the interdomain linker. In the presence of ATP, the linker binds to the edge of the IIA ß-sheet, which structurally connects the linker and the nucleotide-binding site. Thus, a pathway of allosteric communication leads from the NBD nucleotide-binding site to the substrate-binding domain via the interdomain linker.

Fractal Phototherapy in Maximizing Retina and Brain Plasticity.

The neuroplasticity potential is reduced with aging and impairs during neurodegenerative diseases and brain and visual system injuries. This limits the brain's capacity to repair the structure and dynamics of its activity after lesions. Maximization of neuroplasticity is necessary to provide the maximal CNS response to therapeutic intervention and adaptive reorganization of neuronal networks in patients with degenerative pathology and traumatic injury to restore the functional activity of the brain and retina.Considering the fractal geometry and dynamics of the healthy brain and the loss of fractality in neurodegenerative pathology, we suggest that the application of self-similar visual signals with a fractal temporal structure in the stimulation therapy can reactivate the adaptive neuroplasticity and enhance the effectiveness of neurorehabilitation. This proposition was tested in the recent studies. Patients with glaucoma had a statistically significant positive effect of fractal photic therapy on light sensitivity and the perimetric MD index, which shows that methods of fractal stimulation can be a novel nonpharmacological approach to neuroprotective therapy and neurorehabilitation. In healthy rabbits, it was demonstrated that a long-term course of photostimulation with fractal signals does not harm the electroretinogram (ERG) and retina structure. Rabbits with modeled retinal atrophy showed better dynamics of the ERG restoration during daily stimulation therapy for a week in comparison with the controls. Positive changes in the retinal function can indirectly suggest the activation of its adaptive plasticity and the high potential of stimulation therapy with fractal visual stimuli in a nonpharmacological neurorehabilitation, which requires further study.

Delicate balance between functionally required flexibility and aggregation risk in a ß-rich protein.

Susceptibility to aggregation is general to proteins because of the potential for intermolecular interactions between hydrophobic stretches in their amino acid sequences. Protein aggregation has been implicated in several catastrophic diseases, yet we still lack in-depth understanding about how proteins are channeled to this state. Using a predominantly ß-sheet protein whose folding has been explored in detail, cellular retinoic acid-binding protein 1 (CRABP1), as a model, we have tackled the challenge of understanding the links between a protein's natural tendency to fold, 'breathe', and function with its propensity to misfold and aggregate. We identified near-native dynamic species that lead to aggregation and found that inherent structural fluctuations in the native protein, resulting in opening of the ligand-entry portal, expose hydrophobic residues on the most vulnerable aggregation-prone sequences in CRABP1. CRABP1 and related intracellullar lipid-binding proteins have not been reported to aggregate inside cells, and we speculate that the cellular concentration of their open, aggregation-prone conformations is sufficient for ligand binding but below the critical concentration for aggregation. Our finding provides an example of how nature fine-tunes a delicate balance between protein function, conformational variability, and aggregation vulnerability and implies that with the evolutionary requirement for proteins to fold and function, aggregation becomes an unavoidable but controllable risk.

Peptaibol antiamoebin I: spatial structure, backbone dynamics, interaction with bicelles and lipid-protein nanodiscs, and pore formation in context of barrel-stave model.

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. In comparison with other 16-amino acid-residue peptaibols, e.g., zervamicin IIB (Zrv-IIB), Aam-I possesses relatively weak biological and channel-forming activities. In MeOH solution, Aam-I demonstrates fast cooperative transitions between right-handed and left-handed helical conformation of the N-terminal (1-8) region. We studied Aam-I spatial structure and backbone dynamics in the membrane-mimicking environment (DMPC/DHPC bicelles)(1) ) by heteronuclear (1) H,(13) C,(15) N-NMR spectroscopy. Interaction with the bicelles stabilizes the Aam-I right-handed helical conformation retaining significant intramolecular mobility on the ms-µs time scale. Extensive ms-µs dynamics were also detected in the DPC and DHPC micelles and DOPG nanodiscs. In contrast, Zrv-IIB in the DPC micelles demonstrates appreciably lesser mobility on the µs-ms time scale. Titration with Mn(2+) and 16-doxylstearate paramagnetic probes revealed Aam-I binding to the bicelle surface with the N-terminus slightly immersed into hydrocarbon region. Fluctuations of the Aam-I helix between surface-bound and transmembrane (TM) state were observed in the nanodisc membranes formed from the short-chain (diC12 : 0) DLPC/DLPG lipids. All the obtained experimental data are in agreement with the barrel-stave model of TM pore formation, similarly to the mechanism proposed for Zrv-IIB and other peptaibols. The observed extensive intramolecular dynamics explains the relatively low activity of Aam-I.

Caribbean salinity anomalies contributed to variable North Atlantic circulation and climate during the Common Era.

Paleoceanographic reconstructions show that the strength of North Atlantic currents decreased during the Little Ice Age. In contrast, the role of ocean circulation in climate regulation during earlier historical epochs of the Common Era (C.E.) remains unclear. Here, we reconstruct sea surface temperature (SST) and salinity in the Caribbean Basin for the past 1700 years using the isotopic and elemental composition of planktic foraminifera tests. Centennial-scale SST and salinity variations in the Caribbean co-occur with (hydro)climate changes in the Northern Hemisphere and are linked to a North Atlantic SST forcing. Cold phases around 600, 800, and 1400 to 1600 C.E. are characterized by Caribbean salinification and Gulf of Mexico freshening that implies reductions in the strength of North Atlantic surface circulation. We suggest that the associated changes in the meridional salt advection contributed to the historical climate variability of the C.E.

Bouquet Formation Failure in Meiosis of F1 Wheat-Rye Hybrids with Mitotic-Like Division.

Bouquet formation is believed to be involved in initiating homologous chromosome pairings in meiosis. A bouquet is also formed in the absence of chromosome pairing, such as in F1 wheat-rye hybrids. In some hybrids, meiosis is characterized by a single, mitotic-like division that leads to the formation of unreduced gametes. In this study, FISH with the telomere and centromere-specific probe, and immunoFISH with ASY1, CENH3 and rye subtelomere repeat pSc200 were employed to perform a comparative analysis of early meiotic prophase nuclei in four combinations of wheat-rye hybrids. One of these, with disomic rye chromosome 2R, is known to undergo normal meiosis, and here, 78.9% of the meiocytes formed a normal-appearing telomere bouquet and rye subtelomeres clustered in 83.2% of the meiocytes. In three combinations with disomic rye chromosomes 1R, 5R and 6R, known to undergo a single division of meiosis, telomeres clustered in 11.4%, 44.8% and 27.6% of the meiocytes, respectively. In hybrids with chromosome 1R, rye subtelomeres clustered in 12.19% of the meiocytes. In the remaining meiocytes, telomeres and subtelomeres were scattered along the nucleus circumference, forming large and small groups. We conclude that in wheat-rye hybrids with mitotic-like meiosis, chromosome behavior is altered already in the early prophase.

Dynamic interplay between the periplasmic chaperone SurA and the BAM complex in outer membrane protein folding.

Correct folding of outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria depends on delivery of unfolded OMPs to the ß-barrel assembly machinery (BAM). How unfolded substrates are presented to BAM remains elusive, but the major OMP chaperone SurA is proposed to play a key role. Here, we have used hydrogen deuterium exchange mass spectrometry (HDX-MS), crosslinking, in vitro folding and binding assays and computational modelling to show that the core domain of SurA and one of its two PPIase domains are key to the SurA-BAM interaction and are required for maximal catalysis of OMP folding. We reveal that binding causes changes in BAM and SurA conformation and/or dynamics distal to the sites of binding, including at the BamA ß1-ß16 seam. We propose a model for OMP biogenesis in which SurA plays a crucial role in OMP delivery and primes BAM to accept substrates for folding.

Exploring weak, transient protein--protein interactions in crowded in vivo environments by in-cell nuclear magnetic resonance spectroscopy.

Biology relies on functional interplay of proteins in the crowded and heterogeneous environment inside cells, and functional protein interactions are often weak and transient. Thus, methods that preserve these interactions and provide information about them are needed. In-cell nuclear magnetic resonance (NMR) spectroscopy is an attractive method for studying a protein's behavior in cells because it may provide residue-level structural and dynamic information, yet several factors limit the feasibility of protein NMR spectroscopy in cells; among them, slow rotational diffusion has emerged as the most important. In this paper, we seek to elucidate the causes of the dramatically slow protein tumbling in cells and in so doing to gain insight into how the intracellular viscosity and weak, transient interactions modulate protein mobility. To address these questions, we characterized the rotational diffusion of three model globular proteins in Escherichia coli cells using two-dimensional heteronuclear NMR spectroscopy. These proteins have a similar molecular size and globular fold but very different surface properties, and indeed, they show very different rotational diffusion in the E. coli intracellular environment. Our data are consistent with an intracellular viscosity approximately 8 times that of water, too low to be a limiting factor for observation of small globular proteins by in-cell NMR spectroscopy. Thus, we conclude that transient interactions with cytoplasmic components significantly and differentially affect the mobility of proteins and therefore their NMR detectability. Moreover, we suggest that an intricate interplay of total protein charge and hydrophobic interactions plays a key role in regulating these weak intermolecular interactions in cells.

First Virtual International Congress on Cellular and Organismal Stress Responses, November 5-6, 2020.

Members of the Cell Stress Society International (CSSI), Patricija van Oosten-Hawle (University of Leeds, UK), Mehdi Mollapour (SUNY Upstate Medical University, USA), Andrew Truman (University of North Carolina at Charlotte, USA) organized a new virtual meeting format which took place on November 5-6, 2020. The goal of this congress was to provide an international platform for scientists to exchange data and ideas among the Cell Stress and Chaperones community during the Covid-19 pandemic. Here we will highlight the summary of the meeting and acknowledge those who were honored by the CSSI.

Amyloid binding and beyond: a new approach for Alzheimer's disease drug discovery targeting Aßo-PrPC binding and downstream pathways.

Amyloid ß oligomers (Aßo) are the main toxic species in Alzheimer's disease, which have been targeted for single drug treatment with very little success. In this work we report a new approach for identifying functional Aßo binding compounds. A tailored library of 971 fluorine containing compounds was selected by a computational method, developed to generate molecular diversity. These compounds were screened for Aßo binding by a combined 19F and STD NMR technique. Six hits were evaluated in three parallel biochemical and functional assays. Two compounds disrupted Aßo binding to its receptor PrPC in HEK293 cells. They reduced the pFyn levels triggered by Aßo treatment in neuroprogenitor cells derived from human induced pluripotent stem cells (hiPSC). Inhibitory effects on pTau production in cortical neurons derived from hiPSC were also observed. These drug-like compounds connect three of the pillars in Alzheimer's disease pathology, i.e. prion, Aß and Tau, affecting three different pathways through specific binding to Aßo and are, indeed, promising candidates for further development.

Random chromosome distribution in the first meiosis of F1 disomic substitution line 2R(2D) x rye hybrids (ABDR, 4× = 28) occurs without bipolar spindle assembly.

The assembly of the microtubule-based spindle structure in plant meiosis remains poorly understood compared with our knowledge of mitotic spindle formation. One of the approaches in our understanding of microtubule dynamics is to study spindle assembly in meiosis of amphyhaploids. Using immunostaining with phH3Ser10, CENH3 and α-tubulin-specific antibodies, we studied the chromosome distribution and spindle organisation in meiosis of F1 2R(2D)xR wheat-rye hybrids (genome structure ABDR, 4× = 28), as well as in wheat and rye mitosis and meiosis. At the prometaphase of mitosis, spindle assembly was asymmetric; one half of the spindle assembled before the other, with simultaneous chromosome alignment in the spindle mid-zone. At diakinesis in wheat and rye, microtubules formed a pro-spindle which was subsequently disassembled followed by a bipolar spindle assembly. In the first meiosis of hybrids 2R(2D)xR, a bipolar spindle was not found and the kinetochore microtubules distributed the chromosomes. Univalent chromosomes are characterised by a monopolar orientation and maintenance of sister chromatid and centromere cohesion. Presence of bivalents did not affect the formation of a bipolar spindle. Since the central spindle was absent, phragmoplast originates from "interpolar" microtubules generated by kinetochores. Cell plate development occurred with a delay. However, meiocytes in meiosis II contained apparently normal bipolar spindles. Thus, we can conclude that: (1) cohesion maintenance in centromeres and between arms of sister chromatids may negatively affect bipolar spindle formation in the first meiosis; (2) 2R/2D rye/wheat chromosome substitution affects the regulation of the random chromosome distribution in the absence of a bipolar spindle.

Hyperdimensional NMR spectroscopy with nonlinear sampling.

An approach is described for joint interleaved recording, real-time processing, and analysis of NMR data sets. The method employs multidimensional decomposition to find common information in a set of conventional triple-resonance spectra recorded in the nonlinear sampling mode, and builds a model of hyperdimensional (HD) spectrum. While preserving sensitivity per unit of measurement time and allowing for maximal spectral resolution, the approach reduces data collection time on average by 2 orders of magnitude compared to the conventional method. The 7-10 dimensional HD spectrum, which is represented as a set of deconvoluted 1D vectors, is easy to handle and amenable for automated analysis. The method is exemplified by automated assignment for two protein systems of low and high spectral complexity: ubiquitin (globular, 8 kDa) and zetacyt (naturally disordered, 13 kDa). The collection and backbone assignment of the data sets are achieved in real time after approximately 1 and 10 h, respectively. The approach removes the most critical time bottlenecks in data acquisition and analysis. Thus, it can significantly increase the value of NMR spectroscopy in structural biology, for example, in high-throughput structural genomics applications.

Propagation of dynamic changes in barnase upon binding of barstar: an NMR and computational study.

NMR spectroscopy and computer simulations were used to examine changes in chemical shifts and in dynamics of the ribonuclease barnase that result upon binding to its natural inhibitor barstar. Although the spatial structures of free and bound barnase are very similar, binding results in changes of the dynamics of both fast side-chains, as revealed by (2)H relaxation measurements, and NMR chemical shifts in an extended beta-sheet that is located far from the binding interface. Both side-chain dynamics and chemical shifts are sensitive to variations in the ensemble populations of the inter-converting molecular states, which can escape direct structural observation. Molecular dynamics simulations of free barnase and barnase in complex with barstar, as well as a normal mode analysis of barnase using a Gaussian network model, reveal relatively rigid domains that are separated by the extended beta-sheet mentioned above. The observed changes in NMR parameters upon ligation can thus be rationalized in terms of changes in inter-domain dynamics and in populations of exchanging states, without measurable structural changes. This provides an alternative model for the propagation of a molecular response to ligand binding across a protein that is based exclusively on changes in dynamics.

Mitotic phosphorylation regulates Hsp72 spindle localization by uncoupling ATP binding from substrate release.

Hsp72 is a member of the 70-kDa heat shock family of molecular chaperones (Hsp70s) that comprise a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD) connected by a linker that couples the exchange of adenosine diphosphate (ADP) for adenosine triphosphate (ATP) with the release of the protein substrate. Mitotic phosphorylation of Hsp72 by the kinase NEK6 at Thr66 located in the NBD promotes the localization of Hsp72 to the mitotic spindle and is required for efficient spindle assembly and chromosome congression and segregation. We determined the crystal structure of the Hsp72 NBD containing a genetically encoded phosphoserine at position 66. This revealed structural changes that stabilized interactions between subdomains within the NBD. ATP binding to the NBD of unmodified Hsp72 resulted in the release of substrate from the SBD, but phosphorylated Hsp72 retained substrate in the presence of ATP. Mutations that prevented phosphorylation-dependent subdomain interactions restored the connection between ATP binding and substrate release. Thus, phosphorylation of Thr66 is a reversible mechanism that decouples the allosteric connection between nucleotide binding and substrate release, providing further insight into the regulation of the Hsp70 family. We propose that phosphorylation of Hsp72 on Thr66 by NEK6 during mitosis promotes its localization to the spindle by stabilizing its interactions with components of the mitotic spindle.

Binding of intrinsically disordered proteins is not necessarily accompanied by a structural transition to a folded form.

There are a large number of protein domains and even entire proteins, lacking ordered structure under physiological conditions. Intriguingly, a highly flexible, random coil-like conformation is the native and functional state for many proteins known to be involved in cell signaling. An example is a key component of immune signaling, the cytoplasmic region of the T cell receptor zeta subunit. This domain exhibits specific dimerization that is distinct from non-specific aggregation behavior seen in many systems. In this work, we use diffusion and chemical shift mapping NMR data to show that the protein does not undergo a transition between disordered and ordered states upon dimerization. This finding opposes the generally accepted view on the behavior of intrinsically disordered proteins, provides evidence for the existence of specific dimerization interactions for intrinsically disordered protein species and opens a new line of research in this new and quickly developing field.

Allosteric fine-tuning of the conformational equilibrium poises the chaperone BiP for post-translational regulation.

BiP is the only Hsp70 chaperone in the endoplasmic reticulum (ER) and similar to other Hsp70s, its activity relies on nucleotide- and substrate-controllable docking and undocking of its nucleotide-binding domain (NBD) and substrate-binding domain (SBD). However, little is known of specific features of the BiP conformational landscape that tune BiP to its unique tasks and the ER environment. We present methyl NMR analysis of the BiP chaperone cycle that reveals surprising conformational heterogeneity of ATP-bound BiP that distinguishes BiP from its bacterial homologue DnaK. This unusual poise enables gradual post-translational regulation of the BiP chaperone cycle and its chaperone activity by subtle local perturbations at SBD allosteric 'hotspots'. In particular, BiP inactivation by AMPylation of its SBD does not disturb Hsp70 inter-domain allostery and preserves BiP structure. Instead it relies on a redistribution of the BiP conformational ensemble and stabilization the domain-docked conformation in presence of ADP and ATP.