DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity.

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8 that drives self-assembly through π-π stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

The outermost layer of centrosomes, called pericentriolar material (PCM), organizes microtubules for mitotic spindle assembly. The molecular interactions that enable PCM to assemble and resist external forces are poorly understood. Here, we use crosslinking mass spectrometry (XL-MS) to analyze PLK-1-potentiated multimerization of SPD-5, the main PCM scaffold protein in C. elegans. In the unassembled state, SPD-5 exhibits numerous intramolecular crosslinks that are eliminated after phosphorylation by PLK-1. Thus, phosphorylation induces a structural opening of SPD-5 that primes it for assembly. Multimerization of SPD-5 is driven by interactions between multiple dispersed coiled-coil domains. Structural analyses of a phosphorylated region (PReM) in SPD-5 revealed a helical hairpin that dimerizes to form a tetrameric coiled-coil. Mutations within this structure and other interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces, revealing that PCM assembly and strength are interdependent. We propose that PCM size and strength emerge from specific, multivalent coiled-coil interactions between SPD-5 proteins.

DNAJB8 oligomerization is mediated by an aromatic-rich motif that is dispensable for substrate activity.

J-domain protein (JDP) molecular chaperones have emerged as central players that maintain a healthy proteome. The diverse members of the JDP family function as monomers/dimers and a small subset assemble into micron-sized oligomers. The oligomeric JDP members have eluded structural characterization due to their low-complexity, intrinsically disordered middle domains. This in turn, obscures the biological significance of these larger oligomers in protein folding processes. Here, we identified a short, aromatic motif within DNAJB8, that drives self-assembly through pi-pi stacking and determined its X-ray structure. We show that mutations in the motif disrupt DNAJB8 oligomerization in vitro and in cells. DNAJB8 variants that are unable to assemble bind to misfolded tau seeds more specifically and retain capacity to reduce protein aggregation in vitro and in cells. We propose a new model for DNAJB8 function in which the sequences in the low-complexity domains play distinct roles in assembly and substrate activity.

De novo designed Hsp70 activator dissolves intracellular condensates.

Protein quality control (PQC) is carried out in part by the chaperone Hsp70, in concert with adapters of the J-domain protein (JDP) family. The JDPs, also called Hsp40s, are thought to recruit Hsp70 into complexes with specific client proteins. However, the molecular principles regulating this process are not well understood. We describe the de novo design of a set of Hsp70 binding proteins that either inhibited or stimulated Hsp70's ATPase activity; a stimulating design promoted the refolding of denatured luciferase in vitro, similar to native JDPs. Targeting of this design to intracellular condensates resulted in their nearly complete dissolution. The designs inform our understanding of chaperone structure-function relationships and provide a general and modular way to target PQC systems to condensates and other cellular targets.

A central helical hairpin in SPD-5 enables centrosome strength and assembly.

Centrosomes organize microtubules for mitotic spindle assembly and positioning. Forces mediated by these microtubules create tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. How PCM resists these stresses is unclear at the molecular level. Here, we use cross-linking mass spectrometry (XL-MS) to map interactions underlying multimerization of SPD-5, an essential PCM scaffold component in C. elegans . We identified an interaction hotspot in an alpha helical hairpin motif in SPD-5 (a.a. 541-677). XL-MS data, ab initio structural predictions, and mass photometry suggest that this region dimerizes to form a tetrameric coiled-coil. Mutating a helical section (a.a. 610-640) or a single residue (R592) inhibited PCM assembly in embryos. This phenotype was rescued by eliminating microtubule pulling forces, revealing that PCM assembly and material strength are interrelated. We propose that interactions mediated by the helical hairpin strongly bond SPD-5 molecules to each other, thus enabling PCM to assemble fully and withstand stresses generated by microtubules.

Multivalent coiled-coil interactions enable full-scale centrosome assembly and strength.

During mitotic spindle assembly, microtubules generate tensile stresses on pericentriolar material (PCM), the outermost layer of centrosomes. The molecular interactions that enable PCM to assemble rapidly and resist external forces are unknown. Here we use cross-linking mass spectrometry to identify interactions underlying supramolecular assembly of SPD-5, the main PCM scaffold protein in C. elegans . Crosslinks map primarily to alpha helices within the phospho-regulated region (PReM), a long C-terminal coiled-coil, and a series of four N-terminal coiled-coils. PLK-1 phosphorylation of SPD-5 creates new homotypic contacts, including two between PReM and the CM2-like domain, and eliminates numerous contacts in disordered linker regions, thus favoring coiled-coil-specific interactions. Mutations within these interacting regions cause PCM assembly defects that are partly rescued by eliminating microtubule-mediated forces. Thus, PCM assembly and strength are interdependent. In vitro , self-assembly of SPD-5 scales with coiled-coil content, although there is a defined hierarchy of association. We propose that multivalent interactions among coiled-coil regions of SPD-5 build the PCM scaffold and contribute sufficient strength to resist microtubule-mediated forces.

Network of hotspot interactions cluster tau amyloid folds.

Cryogenic electron microscopy has revealed unprecedented molecular insight into the conformations of ß-sheet-rich protein amyloids linked to neurodegenerative diseases. It remains unknown how a protein can adopt a diversity of folds and form multiple distinct fibrillar structures. Here we develop an in silico alanine scan method to estimate the relative energetic contribution of each amino acid in an amyloid assembly. We apply our method to twenty-seven ex vivo and in vitro fibril structural polymorphs of the microtubule-associated protein tau. We uncover networks of energetically important interactions involving amyloid-forming motifs that stabilize the different fibril folds. We evaluate our predictions in cellular and in vitro aggregation assays. Using a machine learning approach, we classify the structures based on residue energetics to identify distinguishing and unifying features. Our energetic profiling suggests that minimal sequence elements control the stability of tau fibrils, allowing future design of protein sequences that fold into unique structures.

Chaperoning shape-shifting tau in disease.

Many neurodegenerative diseases, including Alzheimer's, originate from the conversion of proteins into pathogenic conformations. The microtubule-associated protein tau converts into ß-sheet-rich amyloid conformations, which underlie pathology in over 25 related tauopathies. Structural studies of tau amyloid fibrils isolated from human tauopathy tissues have revealed that tau adopts diverse structural polymorphs, each linked to a different disease. Molecular chaperones play central roles in regulating tau function and amyloid assembly in disease. New data supports the model that chaperones selectively recognize different conformations of tau to limit the accumulation of proteotoxic species. The challenge now is to understand how chaperones influence disease processes across different tauopathies, which will help guide the development of novel conformation-specific diagnostic and therapeutic strategies.

DnaJC7 binds natively folded structural elements in tau to inhibit amyloid formation.

Molecular chaperones, including Hsp70/J-domain protein (JDP) families, play central roles in binding substrates to prevent their aggregation. How JDPs select different conformations of substrates remains poorly understood. Here, we report an interaction between the JDP DnaJC7 and tau that efficiently suppresses tau aggregation in vitro and in cells. DnaJC7 binds preferentially to natively folded wild-type tau, but disease-associated mutants in tau reduce chaperone binding affinity. We identify that DnaJC7 uses a single TPR domain to recognize a ß-turn structural element in tau that contains the 275VQIINK280 amyloid motif. Wild-type tau, but not mutant, ß-turn structural elements can block full-length tau binding to DnaJC7. These data suggest DnaJC7 preferentially binds and stabilizes natively folded conformations of tau to prevent tau conversion into amyloids. Our work identifies a novel mechanism of tau aggregation regulation that can be exploited as both a diagnostic and a therapeutic intervention.

Biophysical properties of a tau seed.

Pathogenesis of tauopathies involves conversion of tau monomer into pathological tau conformers that serve as templates to recruit native tau into growing assemblies. Small soluble tau seeds have been proposed to drive pathological tau assembly in vitro, in cells and in vivo. We have previously described the isolation of monomeric pathogenic tau seeds derived from recombinant samples and tauopathy tissues but in-depth biophysical characterization of these species has not been done. Here we describe a chromatographic method to isolate recombinant soluble tau seeds derived from heparin treatment. We used biochemical and biophysical approaches to show that the seeds are predominantly monomeric and have the capacity to nucleate aggregation of inert forms of tau in vitro and in cells. Finally, we used crosslinking mass spectrometry to identify the topological changes in tau as it converts from an inert state to a pathogenic seed. Future studies will reveal the relationship between soluble seeds and structural polymorphs derived from tauopathies to help diagnose and develop therapeutics targeting specific tauopathies.

Regulatory inter-domain interactions influence Hsp70 recruitment to the DnaJB8 chaperone.

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network.

Tau local structure shields an amyloid-forming motif and controls aggregation propensity.

Tauopathies are neurodegenerative diseases characterized by intracellular amyloid deposits of tau protein. Missense mutations in the tau gene (MAPT) correlate with aggregation propensity and cause dominantly inherited tauopathies, but their biophysical mechanism driving amyloid formation is poorly understood. Many disease-associated mutations localize within tau's repeat domain at inter-repeat interfaces proximal to amyloidogenic sequences, such as 306VQIVYK311. We use cross-linking mass spectrometry, recombinant protein and synthetic peptide systems, in silico modeling, and cell models to conclude that the aggregation-prone 306VQIVYK311 motif forms metastable compact structures with its upstream sequence that modulates aggregation propensity. We report that disease-associated mutations, isomerization of a critical proline, or alternative splicing are all sufficient to destabilize this local structure and trigger spontaneous aggregation. These findings provide a biophysical framework to explain the basis of early conformational changes that may underlie genetic and sporadic tau pathogenesis.