A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

The self-association equilibrium of DNAJA2 regulates its interaction with unfolded substrate proteins and with Hsc70.

J-domain proteins tune the specificity of Hsp70s, engaging them in precise functions. Despite their essential role, the structure and function of many J-domain proteins remain largely unknown. We explore human DNAJA2, finding that it reversibly forms highly-ordered, tubular structures that can be dissociated by Hsc70, the constitutively expressed Hsp70 isoform. Cryoelectron microscopy and mutational studies reveal that different domains are involved in self-association. Oligomer dissociation into dimers potentiates its interaction with unfolded client proteins. The J-domains are accessible to Hsc70 within the tubular structure. They allow binding of closely spaced Hsc70 molecules that could be transferred to the unfolded substrate for its cooperative remodelling, explaining the efficient recovery of DNAJA2-bound clients. The disordered C-terminal domain, comprising the last 52 residues, regulates its holding activity and productive interaction with Hsc70. These in vitro findings suggest that the association equilibrium of DNAJA2 could regulate its interaction with client proteins and Hsc70.

Chaperonin Mechanisms: Multiple and (Mis)Understood?

The chaperonins are ubiquitous and essential nanomachines that assist in protein folding in an ATP-driven manner. They consist of two back-to-back stacked oligomeric rings with cavities in which protein (un)folding can take place in a shielding environment. This review focuses on GroEL from Escherichia coli and the eukaryotic chaperonin-containing t-complex polypeptide 1, which differ considerably in their reaction mechanisms despite sharing a similar overall architecture. Although chaperonins feature in many current biochemistry textbooks after being studied intensively for more than three decades, key aspects of their reaction mechanisms remain under debate and are discussed in this review. In particular, it is unclear whether a universal reaction mechanism operates for all substrates and whether it is passive, i.e., aggregation is prevented but the folding pathway is unaltered, or active. It is also unclear how chaperonin clients are distinguished from nonclients and what are the precise roles of the cofactors with which chaperonins interact.

Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.

In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the Podoviridae family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects Escherichia coli, and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.

T cell asymmetry and metabolic crosstalk can fine-tune immunological synapses.

T cell asymmetry upon specific cell-cell interactions during mammalian immunological synapse (IS) contacts requires mammalian target of rapamycin complex (mTORC) activation and chaperones, such as the eukaryotic chaperonin containing TCP1 (CCT) for protein synthesis and folding. This mechanism can control cytoskeleton dynamics, and regulate mitochondrial fate, respiration, and metabolic rates, ultimately underlying cell reprogramming events that are relevant for CD4+ T cell functional outcomes.

Folding for the Immune Synapse: CCT Chaperonin and the Cytoskeleton.

Lymphocytes rearrange their shape, membrane receptors and organelles during cognate contacts with antigen-presenting cells (APCs). Activation of T cells by APCs through pMHC-TCR/CD3 interaction (peptide-major histocompatibility complex-T cell receptor/CD3 complexes) involves different steps that lead to the reorganization of the cytoskeleton and organelles and, eventually, activation of nuclear factors allowing transcription and ultimately, replication and cell division. Both the positioning of the lymphocyte centrosome in close proximity to the APC and the nucleation of a dense microtubule network beneath the plasma membrane from the centrosome support the T cell's intracellular polarity. Signaling from the TCR is facilitated by this traffic, which constitutes an important pathway for regulation of T cell activation. The coordinated enrichment upon T cell stimulation of the chaperonin CCT (chaperonin-containing tailless complex polypeptide 1; also termed TRiC) and tubulins at the centrosome area support polarized tubulin polymerization and T cell activation. The proteasome is also enriched in the centrosome of activated T cells, providing a mechanism to balance local protein synthesis and degradation. CCT assists the folding of proteins coming from de novo synthesis, therefore favoring mRNA translation. The functional role of this chaperonin in regulating cytoskeletal composition and dynamics at the immune synapse is discussed.

Chaperonins: Nanocarriers with Biotechnological Applications.

Chaperonins are molecular chaperones found in all kingdoms of life, and as such they assist in the folding of other proteins. Structurally, chaperonins are cylinders composed of two back-to-back rings, each of which is an oligomer of ~60-kDa proteins. Chaperonins are found in two main conformations, one in which the cavity is open and ready to recognise and trap unfolded client proteins, and a "closed" form in which folding takes place. The conspicuous properties of this structure (a cylinder containing a cavity that allows confinement) and the potential to control its closure and aperture have inspired a number of nanotechnological applications that will be described in this review.

The cochaperone CHIP marks Hsp70- and Hsp90-bound substrates for degradation through a very flexible mechanism.

Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST). Both ternary complexes (Hsp70:p53-TMGST:CHIP and Hsp90:p53-TMGST:CHIP) ubiquitinated the substrate at a higher efficiency than in the absence of the chaperones. The 3D structures of the two complexes, obtained using a combination of cryoelectron microscopy and crosslinking mass spectrometry, showed the substrate located between the chaperone and the cochaperone, suggesting a ubiquitination mechanism in which the chaperone-bound substrate is presented to CHIP. These complexes are inherently flexible, which is important for the ubiquitination process.

Hsp70 chaperone: a master player in protein homeostasis.

Protein homeostasis (proteostasis) is an essential pillar for correct cellular function. Impairments in proteostasis are encountered both in aging and in several human disease conditions. Molecular chaperones are important players for proteostasis; in particular, heat shock protein 70 (Hsp70) has an essential role in protein folding, disaggregation, and degradation. We have recently proposed a model for Hsp70 functioning as a "multiple socket". In the model, Hsp70 provides a physical platform for the binding of client proteins, other chaperones, and cochaperones. The final fate of the client protein is dictated by the set of Hsp70 interactions that occur in a given cellular context. Obtaining structural information of the different Hsp70-based protein complexes will provide valuable knowledge to understand the functional mechanisms behind the master role of Hsp70 in proteostasis. We additionally evaluate some of the challenges for attaining high-resolution structures of such complexes.

The chaperonin CCT promotes the formation of fibrillar aggregates of γ-tubulin.

The type II chaperonin CCT is involved in the prevention of the pathogenesis of numerous human misfolding disorders, as it sequesters misfolded proteins, blocks their aggregation and helps them to achieve their native state. In addition, it has been reported that CCT can prevent the toxicity of non-client amyloidogenic proteins by the induction of non-toxic aggregates, leading to new insight in chaperonin function as an aggregate remodeling factor. Here we add experimental evidence to this alternative mechanism by which CCT actively promotes the formation of conformationally different aggregates of γ-tubulin, a non-amyloidogenic CCT client protein, which are mediated by specific CCT-γ-tubulin interactions. The in vitro-induced aggregates were in some cases long fiber polymers, which compete with the amorphous aggregates. Direct injection of unfolded purified γ-tubulin into single-cell zebra fish embryos allowed us to relate this in vitro activity with the in vivo formation of intracellular aggregates. Injection of a CCT-binding deficient γ-tubulin mutant dramatically diminished the size of the intracellular aggregates, increasing the toxicity of the misfolded protein. These results point to CCT having a role in the remodeling of aggregates, constituting one of its many functions in cellular proteostasis.

Domain topology of human Rasal.

Rasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BP and Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BP and Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

Hsp70 - a master regulator in protein degradation.

Proteostasis, the controlled balance of protein synthesis, folding, assembly, trafficking and degradation, is a paramount necessity for cell homeostasis. Impaired proteostasis is a hallmark of ageing and of many human diseases. Molecular chaperones are essential for proteostasis in eukaryotic cells, and their function has traditionally been linked to protein folding, assembly and disaggregation. More recent findings suggest that chaperones also contribute to key steps in protein degradation. In particular, Hsp70 has an essential role in substrate degradation through the ubiquitin-proteasome system, as well as through different autophagy pathways. Accumulated knowledge suggests that the fate of an Hsp70 substrate is dictated by the combination of partners (cochaperones and other chaperones) that interact with Hsp70 in a given cell context.

Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery.

Molecular chaperones: functional mechanisms and nanotechnological applications.

Molecular chaperones are a group of proteins that assist in protein homeostasis. They not only prevent protein misfolding and aggregation, but also target misfolded proteins for degradation. Despite differences in structure, all types of chaperones share a common general feature, a surface that recognizes and interacts with the misfolded protein. This and other, more specialized properties can be adapted for various nanotechnological purposes, by modification of the original biomolecules or by de novo design based on artificial structures.

Dynamics, flexibility, and allostery in molecular chaperonins.

The chaperonins are a family of molecular chaperones present in all three kingdoms of life. They are classified into Group I and Group II. Group I consists of the bacterial variants (GroEL) and the eukaryotic ones from mitochondria and chloroplasts (Hsp60), while Group II consists of the archaeal (thermosomes) and eukaryotic cytosolic variants (CCT or TRiC). Both groups assemble into a dual ring structure, with each ring providing a protective folding chamber for nascent and denatured proteins. Their functional cycle is powered by ATP binding and hydrolysis, which drives a series of structural rearrangements that enable encapsulation and subsequent release of the substrate protein. Chaperonins have elaborate allosteric mechanisms to regulate their functional cycle. Long-range negative cooperativity between the two rings ensures alternation of the folding chambers. Positive intra-ring cooperativity, which facilitates concerted conformational transitions within the protein subunits of one ring, has only been demonstrated for Group I chaperonins. In this review, we describe our present understanding of the underlying mechanisms and the structure-function relationships in these complex protein systems with a particular focus on the structural dynamics, allostery, and associated conformational rearrangements.

The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism.

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-ß-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-ß-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-ß-tubulin interface that is caused by a steric interaction between ß-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.

Structural characterization of toxic oligomers that are kinetically trapped during α-synuclein fibril formation.

We describe the isolation and detailed structural characterization of stable toxic oligomers of α-synuclein that have accumulated during the process of amyloid formation. Our approach has allowed us to identify distinct subgroups of oligomers and to probe their molecular architectures by using cryo-electron microscopy (cryoEM) image reconstruction techniques. Although the oligomers exist in a range of sizes, with different extents and nature of ß-sheet content and exposed hydrophobicity, they all possess a hollow cylindrical architecture with similarities to certain types of amyloid fibril, suggesting that the accumulation of at least some forms of amyloid oligomers is likely to be a consequence of very slow rates of rearrangement of their ß-sheet structures. Our findings reveal the inherent multiplicity of the process of protein misfolding and the key role the ß-sheet geometry acquired in the early stages of the self-assembly process plays in dictating the kinetic stability and the pathological nature of individual oligomeric species.

Arc is a flexible modular protein capable of reversible self-oligomerization.

The immediate early gene product Arc (activity-regulated cytoskeleton-associated protein) is posited as a master regulator of long-term synaptic plasticity and memory. However, the physicochemical and structural properties of Arc have not been elucidated. In the present study, we expressed and purified recombinant human Arc (hArc) and performed the first biochemical and biophysical analysis of hArc's structure and stability. Limited proteolysis assays and MS analysis indicate that hArc has two major domains on either side of a central more disordered linker region, consistent with in silico structure predictions. hArc's secondary structure was estimated using CD, and stability was analysed by CD-monitored thermal denaturation and differential scanning fluorimetry (DSF). Oligomerization states under different conditions were studied by dynamic light scattering (DLS) and visualized by AFM and EM. Biophysical analyses show that hArc is a modular protein with defined secondary structure and loose tertiary structure. hArc appears to be pyramid-shaped as a monomer and is capable of reversible self-association, forming large soluble oligomers. The N-terminal domain of hArc is highly basic, which may promote interaction with cytoskeletal structures or other polyanionic surfaces, whereas the C-terminal domain is acidic and stabilized by ionic conditions that promote oligomerization. Upon binding of presenilin-1 (PS1) peptide, hArc undergoes a large structural change. A non-synonymous genetic variant of hArc (V231G) showed properties similar to the wild-type (WT) protein. We conclude that hArc is a flexible multi-domain protein that exists in monomeric and oligomeric forms, compatible with a diverse, hub-like role in plasticity-related processes.

Modulation of the chaperone DnaK allosterism by the nucleotide exchange factor GrpE.

Hsp70 chaperones comprise two domains, the nucleotide-binding domain (Hsp70NBD), responsible for structural and functional changes in the chaperone, and the substrate-binding domain (Hsp70SBD), involved in substrate interaction. Substrate binding and release in Hsp70 is controlled by the nucleotide state of DnaKNBD, with ATP inducing the open, substrate-receptive DnaKSBD conformation, whereas ADP forces its closure. DnaK cycles between the two conformations through interaction with two cofactors, the Hsp40 co-chaperones (DnaJ in Escherichia coli) induce the ADP state, and the nucleotide exchange factors (GrpE in E. coli) induce the ATP state. X-ray crystallography showed that the GrpE dimer is a nucleotide exchange factor that works by interaction of one of its monomers with DnaKNBD. DnaKSBD location in this complex is debated; there is evidence that it interacts with the GrpE N-terminal disordered region, far from DnaKNBD. Although we confirmed this interaction using biochemical and biophysical techniques, our EM-based three-dimensional reconstruction of the DnaK-GrpE complex located DnaKSBD near DnaKNBD. This apparent discrepancy between the functional and structural results is explained by our finding that the tail region of the GrpE dimer in the DnaK-GrpE complex bends and its tip contacts DnaKSBD, whereas the DnaKNBD-DnaKSBD linker contacts the GrpE helical region. We suggest that these interactions define a more complex role for GrpE in the control of DnaK function.