Structural transitions in Orb2 prion-like domain relevant for functional aggregation in memory consolidation.

The recent structural elucidation of ex vivo Drosophila Orb2 fibrils revealed a novel amyloid formed by interdigitated Gln and His residue side chains belonging to the prion-like domain. However, atomic-level details on the conformational transitions associated with memory consolidation remain unknown. Here, we have characterized the nascent conformation and dynamics of the prion-like domain (PLD) of Orb2A using a nonconventional liquid-state NMR spectroscopy strategy based on 13C detection to afford an essentially complete set of 13Cα, 13Cß, 1Hα, and backbone 13CO and 15N assignments. At pH 4, where His residues are protonated, the PLD is disordered and flexible, except for a partially populated α-helix spanning residues 55-60, and binds RNA oligos, but not divalent cations. At pH 7, in contrast, His residues are predominantly neutral, and the Q/H segments adopt minor populations of helical structure, show decreased mobility and start to self-associate. At pH 7, the His residues do not bind RNA or Ca2+, but do bind Zn2+, which promotes further association. These findings represent a remarkable case of structural plasticity, based on which an updated model for Orb2A functional amyloidogenesis is suggested.

Mechanistic Insights into the Role of Molecular Chaperones in Protein Misfolding Diseases: From Molecular Recognition to Amyloid Disassembly.

Age-dependent alterations in the proteostasis network are crucial in the progress of prevalent neurodegenerative diseases, such as Alzheimer's, Parkinson's, or amyotrophic lateral sclerosis, which are characterized by the presence of insoluble protein deposits in degenerating neurons. Because molecular chaperones deter misfolded protein aggregation, regulate functional phase separation, and even dissolve noxious aggregates, they are considered major sentinels impeding the molecular processes that lead to cell damage in the course of these diseases. Indeed, members of the chaperome, such as molecular chaperones and co-chaperones, are increasingly recognized as therapeutic targets for the development of treatments against degenerative proteinopathies. Chaperones must recognize diverse toxic clients of different orders (soluble proteins, biomolecular condensates, organized protein aggregates). It is therefore critical to understand the basis of the selective chaperone recognition to discern the mechanisms of action of chaperones in protein conformational diseases. This review aimed to define the selective interplay between chaperones and toxic client proteins and the basis for the protective role of these interactions. The presence and availability of chaperone recognition motifs in soluble proteins and in insoluble aggregates, both functional and pathogenic, are discussed. Finally, the formation of aberrant (pro-toxic) chaperone complexes will also be disclosed.

ASC Pyrin Domain Self-associates and Binds NLRP3 Protein Using Equivalent Binding Interfaces.

Death domain superfamily members typically act as adaptors mediating in the assembly of supramolecular complexes with critical apoptosis and inflammation functions. These modular proteins consist of death domains, death effector domains, caspase recruitment domains, and pyrin domains (PYD). Despite the high structural similarity among them, only homotypic interactions participate in complex formation, suggesting that subtle factors differentiate each interaction type. It is thus critical to identify these factors as an essential step toward the understanding of the molecular basis of apoptosis and inflammation. The proteins apoptosis-associated speck-like protein containing a CARD (ASC) and NLRP3 play key roles in the regulation of apoptosis and inflammation through self-association and protein-protein interactions mediated by their PYDs. To better understand the molecular basis of their function, we have characterized ASC and NLRP3 PYD self-association and their intermolecular interaction by solution NMR spectroscopy and analytical ultracentrifugation. We found that ASC self-associates and binds NLRP3 PYD through equivalent protein regions, with higher binding affinity for the latter. These regions are located at opposite sides of the protein allowing multimeric complex formation previously shown in ASC PYD fibril assemblies. We show that NLRP3 PYD coexists in solution as a monomer and highly populated large-order oligomerized species. Despite this, we determined its monomeric three-dimensional solution structure by NMR and characterized its binding to ASC PYD. Using our novel structural data, we propose molecular models of ASC·ASC and ASC·NLRP3 PYD early supramolecular complexes, providing new insights into the molecular mechanisms of inflammasome and apoptosis signaling.

Structure of Monomeric Transthyretin Carrying the Clinically Important T119M Mutation.

Mutations in the protein transthyretin can cause as well as protect individuals from transthyretin amyloidosis, an incurable fatal inherited disease. Little is known, however, about the structural basis of pathogenic and clinically protective transthyretin mutants. Here we determined the solution structure of a transthyretin monomer that carries the clinically important T119M mutation. The structure displays a non-native arrangement that is distinct from all known structures of transthyretin and highlights the importance of high-resolution studies in solution for understanding molecular processes that lead to amyloid diseases.

Common features at the start of the neurodegeneration cascade.

Amyloidogenic neurodegenerative diseases are incurable conditions with high social impact that are typically caused by specific, largely disordered proteins. However, the underlying molecular mechanism remains elusive to established techniques. A favored hypothesis postulates that a critical conformational change in the monomer (an ideal therapeutic target) in these "neurotoxic proteins" triggers the pathogenic cascade. We use force spectroscopy and a novel methodology for unequivocal single-molecule identification to demonstrate a rich conformational polymorphism in the monomer of four representative neurotoxic proteins. This polymorphism strongly correlates with amyloidogenesis and neurotoxicity: it is absent in a fibrillization-incompetent mutant, favored by familial-disease mutations and diminished by a surprisingly promiscuous inhibitor of the critical monomeric ß-conformational change, neurotoxicity, and neurodegeneration. Hence, we postulate that specific mechanostable conformers are the cause of these diseases, representing important new early-diagnostic and therapeutic targets. The demonstrated ability to inhibit the conformational heterogeneity of these proteins by a single pharmacological agent reveals common features in the monomer and suggests a common pathway to diagnose, prevent, halt, or reverse multiple neurodegenerative diseases.

Alternative low-populated conformations prompt phase transitions in polyalanine repeat expansions.

Abnormal trinucleotide repeat expansions alter protein conformation causing malfunction and contribute to a significant number of incurable human diseases. Scarce structural insights available on disease-related homorepeat expansions hinder the design of effective therapeutics. Here, we present the dynamic structure of human PHOX2B C-terminal fragment, which contains the longest polyalanine segment known in mammals. The major α-helical conformation of the polyalanine tract is solely extended by polyalanine expansions in PHOX2B, which are responsible for most congenital central hypoventilation syndrome cases. However, polyalanine expansions in PHOX2B additionally promote nascent homorepeat conformations that trigger length-dependent phase transitions into solid condensates that capture wild-type PHOX2B. Remarkably, HSP70 and HSP90 chaperones specifically seize PHOX2B alternative conformations preventing phase transitions. The precise observation of emerging polymorphs in expanded PHOX2B postulates unbalanced phase transitions as distinct pathophysiological mechanisms in homorepeat expansion diseases, paving the way towards the search of therapeutics modulating biomolecular condensates in central hypoventilation syndrome.

Fused in sarcoma undergoes cold denaturation: Implications for phase separation.

The mediation of liquid-liquid phase separation (LLPS) for fused in sarcoma (FUS) protein is generally attributed to the low-complexity, disordered domains and is enhanced at low temperature. The role of FUS folded domains on the LLPS process remains relatively unknown since most studies are mainly based on fragmented FUS domains. Here, we investigate the effect of metabolites on full-length (FL) FUS LLPS using turbidity assays and differential interference contrast (DIC) microscopy, and explore the behavior of the folded domains by nuclear magnetic resonance (NMR) spectroscopy. FL FUS LLPS is maximal at low concentrations of glucose and glutamate, moderate concentrations of NaCl, Zn2+ , and Ca2+ and at the isoelectric pH. The FUS RNA recognition motif (RRM) and zinc-finger (ZnF) domains are found to undergo cold denaturation above 0°C at a temperature that is determined by the conformational stability of the ZnF domain. Cold unfolding exposes buried nonpolar residues that can participate in LLPS-promoting hydrophobic interactions. Therefore, these findings constitute the first evidence that FUS globular domains may have an active role in LLPS under cold stress conditions and in the assembly of stress granules, providing further insight into the environmental regulation of LLPS.

Metamorphism in TDP-43 prion-like domain determines chaperone recognition.

The RNA binding protein TDP-43 forms cytoplasmic inclusions via its C-terminal prion-like domain in several neurodegenerative diseases. Aberrant TDP-43 aggregation arises upon phase de-mixing and transitions from liquid to solid states, following still unknown structural conversions which are primed by oxidative stress and chaperone inhibition. Despite the well-established protective roles for molecular chaperones against protein aggregation pathologies, knowledge on the determinants of chaperone recognition in disease-related prions is scarce. Here we show that chaperones and co-chaperones primarily recognize the structured elements in TDP-43´s prion-like domain. Significantly, while HSP70 and HSP90 chaperones promote TDP-43 phase separation, co-chaperones from the three classes of the large human HSP40 family (namely DNAJA2, DNAJB1, DNAJB4 and DNAJC7) show strikingly different effects on TDP-43 de-mixing. Dismantling of the second helical element in TDP-43 prion-like domain by methionine sulfoxidation impacts phase separation and amyloid formation, abrogates chaperone recognition and alters phosphorylation by casein kinase-1δ. Our results show that metamorphism in the post-translationally modified TDP-43 prion-like domain encodes determinants that command mechanisms with major relevance in disease.

Unequivocal single-molecule force spectroscopy of proteins by AFM using pFS vectors.

Nanomechanical analysis of proteins by single-molecule force spectroscopy based on atomic force microscopy is increasingly being used to investigate the inner workings of mechanical proteins and substrate proteins of unfoldase machines as well as to gain new insight into the process of protein folding. However, such studies are hindered by a number of technical problems, including the noise of the proximal region, ambiguous single-molecule identification, as well as difficulties in protein expression/folding and full-length purification. To overcome these major drawbacks in protein nanomechanics, we designed a family of cloning/expression vectors, termed pFS (plasmid for force spectroscopy), that essentially has an unstructured region to surmount the noisy proximal region, a homomeric polyprotein marker, a carrier to mechanically protect the protein of interest (only the pFS-2 version) that also acts as a reporter, and two purification tags. pFS-2 enables the unambiguous analysis of proteins with low mechanical stability or/and complex force spectra, such as the increasingly abundant class of intrinsically disordered proteins, which are hard to characterize by traditional bulk techniques and have important biological and clinical implications. The advantages, applications, and potential of this ready-to-go system are illustrated through the analysis of representative proteins.

Mechanical properties of ß-catenin revealed by single-molecule experiments.

ß-catenin is a central component of the adaptor complex that links cadherins to the actin cytoskeleton in adherens junctions and thus, it is a good candidate to sense and transmit mechanical forces to trigger specific changes inside the cell. To fully understand its molecular physiology, we must first investigate its mechanical role in mechanotransduction within the cadherin system. We have studied the mechanical response of ß-catenin to stretching using single-molecule force spectroscopy and molecular dynamics. Unlike most proteins analyzed to date, which have a fixed mechanical unfolding pathway, the ß-catenin armadillo repeat region (ARM) displays low mechanostability and multiple alternative unfolding pathways that seem to be modulated by its unstructured termini. These results are supported by steered molecular dynamics simulations, which also predict its mechanical stabilization and unfolding pathway restrictions when the contiguous α-helix of the C-terminal unstructured region is included. Furthermore, simulations of the ARM/E-cadherin cytosolic tail complex emulating the most probable stress geometry occurring in vivo show a mechanical stabilization of the interaction whose magnitude correlates with the length of the stretch of the cadherin cytosolic tail that is in contact with the ARM region.

Nanomechanics of the cadherin ectodomain: "canalization" by Ca2+ binding results in a new mechanical element.

Cadherins form a large family of calcium-dependent cell-cell adhesion receptors involved in development, morphogenesis, synaptogenesis, differentiation, and carcinogenesis through signal mechanotransduction using an adaptor complex that connects them to the cytoskeleton. However, the molecular mechanisms underlying mechanotransduction through cadherins remain unknown, although their extracellular region (ectodomain) is thought to be critical in this process. By single molecule force spectroscopy, molecular dynamics simulations, and protein engineering, here we have directly examined the nanomechanics of the C-cadherin ectodomain and found it to be strongly dependent on the calcium concentration. In the presence of calcium, the ectodomain extends through a defined ("canalized") pathway that involves two mechanical resistance elements: a mechanical clamp from the cadherin domains and a novel mechanostable component from the interdomain calcium-binding regions ("calcium rivet") that is abolished by magnesium replacement and in a mutant intended to impede calcium coordination. By contrast, in the absence of calcium, the mechanical response of the ectodomain becomes largely "decanalized" and destabilized. The cadherin ectodomain may therefore behave as a calcium-switched "mechanical antenna" with very different mechanical responses depending on calcium concentration (which would affect its mechanical integrity and force transmission capability). The versatile mechanical design of the cadherin ectodomain and its dependence on extracellular calcium facilitate a variety of mechanical responses that, we hypothesize, could influence the various adhesive properties mediated by cadherins in tissue morphogenesis, synaptic plasticity, and disease. Our work represents the first step toward the mechanical characterization of the cadherin system, opening the door to understanding the mechanical bases of its mechanotransduction.

On the remarkable mechanostability of scaffoldins and the mechanical clamp motif.

Protein mechanostability is a fundamental biological property that can only be measured by single-molecule manipulation techniques. Such studies have unveiled a variety of highly mechanostable modules (mainly of the Ig-like, beta-sandwich type) in modular proteins subjected to mechanical stress from the cytoskeleton and the metazoan cell-cell interface. Their mechanostability is often attributed to a "mechanical clamp" of secondary structure (a patch of backbone hydrogen bonds) fastening their ends. Here we investigate the nanomechanics of scaffoldins, an important family of scaffolding proteins that assembles a variety of cellulases into the so-called cellulosome, a microbial extracellular nanomachine for cellulose adhesion and degradation. These proteins anchor the microbial cell to cellulose substrates, which makes their connecting region likely to be subjected to mechanical stress. By using single-molecule force spectroscopy based on atomic force microscopy, polyprotein engineering, and computer simulations, here we show that the cohesin I modules from the connecting region of cellulosome scaffoldins are the most robust mechanical proteins studied experimentally or predicted from the entire Protein Data Bank. The mechanostability of the cohesin modules studied correlates well with their mechanical kinetic stability but not with their thermal stability, and it is well predicted by computer simulations, even coarse-grained. This extraordinary mechanical stability is attributed to 2 mechanical clamps in tandem. Our findings provide the current upper limit of protein mechanostability and establish shear mechanical clamps as a general structural/functional motif widespread in proteins putatively subjected to mechanical stress. These data have important implications for the scaffoldin physiology and for protein design in biotechnology and nanotechnology.

Global Structure of the Intrinsically Disordered Protein Tau Emerges from Its Local Structure.

The paradigmatic disordered protein tau plays an important role in neuronal function and neurodegenerative diseases. To disentangle the factors controlling the balance between functional and disease-associated conformational states, we build a structural ensemble of the tau K18 fragment containing the four pseudorepeat domains involved in both microtubule binding and amyloid fibril formation. We assemble 129-residue-long tau K18 chains with atomic detail from an extensive fragment library constructed with molecular dynamics simulations. We introduce a reweighted hierarchical chain growth (RHCG) algorithm that integrates experimental data reporting on the local structure into the assembly process in a systematic manner. By combining Bayesian ensemble refinement with importance sampling, we obtain well-defined ensembles and overcome the problem of exponentially varying weights in the integrative modeling of long-chain polymeric molecules. The resulting tau K18 ensembles capture nuclear magnetic resonance (NMR) chemical shift and J-coupling measurements. Without further fitting, we achieve very good agreement with measurements of NMR residual dipolar couplings. The good agreement with experimental measures of global structure such as single-molecule Förster resonance energy transfer (FRET) efficiencies is improved further by ensemble refinement. By comparing wild-type and mutant ensembles, we show that pathogenic single-point P301L, P301S, and P301T mutations shift the population from the turn-like conformations of the functional microtubule-bound state to the extended conformations of disease-associated tau fibrils. RHCG thus provides us with an atomically detailed view of the population equilibrium between functional and aggregation-prone states of tau K18, and demonstrates that global structural characteristics of this intrinsically disordered protein emerge from its local structure.

Molecular mechanism for the synchronized electrostatic coacervation and co-aggregation of alpha-synuclein and tau.

Amyloid aggregation of α-synuclein (αS) is the hallmark of Parkinson's disease and other synucleinopathies. Recently, Tau protein, generally associated with Alzheimer's disease, has been linked to αS pathology and observed to co-localize in αS-rich disease inclusions, although the molecular mechanisms for the co-aggregation of both proteins remain elusive. We report here that αS phase-separates into liquid condensates by electrostatic complex coacervation with positively charged polypeptides such as Tau. Condensates undergo either fast gelation or coalescence followed by slow amyloid aggregation depending on the affinity of αS for the poly-cation and the rate of valence exhaustion of the condensate network. By combining a set of advanced biophysical techniques, we have been able to characterize αS/Tau liquid-liquid phase separation and identified key factors that lead to the formation of hetero-aggregates containing both proteins in the interior of the liquid protein condensates.

Do polyproline II helix associations modulate biomolecular condensates?

Biomolecular condensates are microdroplets that form inside cells and serve to selectively concentrate proteins, RNAs and other molecules for a variety of physiological functions, but can contribute to cancer, neurodegenerative diseases and viral infections. The formation of these condensates is driven by weak, transient interactions between molecules. These weak associations can operate at the level of whole protein domains, elements of secondary structure or even moieties composed of just a few atoms. Different types of condensates do not generally combine to form larger microdroplets, suggesting that each uses a distinct class of attractive interactions. Here, we address whether polyproline II (PPII) helices mediate condensate formation. By combining with PPII-binding elements such as GYF, WW, profilin, SH3 or OCRE domains, PPII helices help form lipid rafts, nuclear speckles, P-body-like neuronal granules, enhancer complexes and other condensates. The number of PPII helical tracts or tandem PPII-binding domains can strongly influence condensate stability. Many PPII helices have a low content of proline residues, which hinders their identification. Recently, we characterized the NMR spectral properties of a Gly-rich, Pro-poor protein composed of six PPII helices. Based on those results, we predicted that many Gly-rich segments may form PPII helices and interact with PPII-binding domains. This prediction is being tested and could join the palette of verified interactions contributing to biomolecular condensate formation.

Molecular basis of the interaction of Hsp90 with its co-chaperone Hop.

The heat shock protein (Hsp) Hsp90 is one of the most abundant proteins in the cell. It controls the functional turnover of proteins being involved in protein folding, refolding, transport as well as protein degradation. Co-chaperones influence Hsp90's activity in different ways, among which the Hsp organizing protein (Hop) was found to inhibit its ATP hydrolysis upon binding. Despite the availability of a number of studies investigating the Hsp90:Hop complex, several aspects of the Hsp90:Hop interaction have remained unresolved. Here, we employed a combinatory approach comprising native polyacrylamide gel electrophoresis, isothermal titration calorimetry, multiangle light scattering, isothermal titration calorimetry, small-angle X-ray scattering, dynamic light scattering, and nuclear magnetic resonance, spectroscopy to obtain a comprehensive picture about the human Hsp90ß:Hop association in solution. Our data show that only one Hop molecule binds the Hsp90ß dimer, Hop can interact with the open and closed state of Hsp90ß, and Hop's TPR2A-2B domains determine the affinity for Hsp90's C-terminal and middle domain, whereby the interaction with the C-terminal domain of Hsp90ß is sufficient to induce an allosteric conformational change between the two Hsp90ß monomers in the Hsp902 :Hop1 complex. Together, this study highlights the important role of the co-chaperone Hop in reorganizing Hsp90 for efficient client loading.

Conformational Priming of RepA-WH1 for Functional Amyloid Conversion Detected by NMR Spectroscopy.

How proteins with a stable globular fold acquire the amyloid state is still largely unknown. RepA, a versatile plasmidic DNA binding protein from Pseudomonas savastanoi, is functional as a transcriptional repressor or as an initiator or inhibitor of DNA replication, the latter via assembly of an amyloidogenic oligomer. Its N-terminal domain (WH1) is responsible for discrimination between these functional abilities by undergoing insufficiently understood structural changes. RepA-WH1 is a stable dimer whose conformational dynamics had not been explored. Here, we have studied it through NMR {1H}-15N relaxation and H/D exchange kinetics measurements. The N- and the C-terminal α-helices, and the internal amyloidogenic loop, are partially unfolded in solution. S4-indigo, a small inhibitor of RepA-WH1 amyloidogenesis, binds to and tethers the N-terminal α-helix to a ß-hairpin that is involved in dimerization, thus providing evidence for a priming role of fraying ends and dimerization switches in the amyloidogenesis of folded proteins.

RNA binding proteins: Diversity from microsurgeons to cowboys.

The conformation and mechanism of proteins that degrade and bind RNA, which has provided key insights into post-transcriptional gene regulation, is explored here. During the twentieth century's last decades, the characterization of ribonucleases and RNA binding domains revealed the diversity of their reaction mechanisms and modes of RNA recognition, and the bases of protein folding, substrate specificity and binding affinity. More recent research showed how these domains combine through oligomerization or genetic recombination to create larger proteins with highly specific and readily programmable ribonucleolytic activity. In the last 15 years, the study of the capacity of proteins, usually disordered, to pool RNAs into discrete, non-aqueous microdroplets to facilitate their transport, modification and degradation - analogous to cowboys herding cattle - has advanced our comprehension of gene expression. Finally, the current uses of RNA binding proteins and the future applications of protein/RNA microdroplets are highlighted.

Dynamic Aha1 co-chaperone binding to human Hsp90.

Hsp90 is an essential chaperone that requires large allosteric changes to determine its ATPase activity and client binding. The co-chaperone Aha1, which is the major ATPase stimulator in eukaryotes, is important for regulation of Hsp90's allosteric timing. Little is known, however, about the structure of the Hsp90/Aha1 complex. Here, we characterize the solution structure of unmodified human Hsp90/Aha1 complex using NMR spectroscopy. We show that the 214-kDa complex forms by a two-step binding mechanism and adopts multiple conformations in the absence of nucleotide. Aha1 induces structural changes near Hsp90's nucleotide-binding site, providing a basis for its ATPase-enhancing activity. Our data reveal important aspects of this pivotal chaperone/co-chaperone interaction and emphasize the relevance of characterizing dynamic chaperone structures in solution.

Nanomechanics of tip-link cadherins.

Hearing and balance rely on the transduction of mechanical stimuli arising from sound waves or head movements into electrochemical signals. This archetypal mechanoelectrical transduction process occurs in the hair-cell stereocilia of the inner ear, which experience continuous oscillations driven by undulations in the endolymph in which they are immersed. The filamentous structures called tip links, formed by an intertwined thread composed of an heterotypic complex of cadherin 23 and protocadherin 15 ectodomain dimers, connect each stereocilium to the tip of the lower sterocilium, and must maintain their integrity against continuous stimulatory deflections. By using single molecule force spectroscopy, here we demonstrate that in contrast to the case of classical cadherins, tip-link cadherins are mechanoresilient structures even at the exceptionally low Ca2+ concentration of the endolymph. We also show that the D101G deafness point mutation in cadherin 23, which affects a Ca2+ coordination site, exhibits an altered mechanical phenotype at the physiological Ca2+ concentration. Our results show a remarkable case of functional adaptation of a protein's nanomechanics to extremely low Ca2+ concentrations and pave the way to a full understanding of the mechanotransduction mechanism mediated by auditory cadherins.