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Amyloid deposition of the microtubule-associated protein tau is associated with neurodegenerative diseases. In frontotemporal dementia with abnormal tau (FTD-tau), missense mutations in tau enhance its aggregation propensity. Here we describe the structural mechanism for how an FTD-tau S320F mutation drives spontaneous aggregation, integrating data from in vitro, in silico and cellular experiments. We find that S320F stabilizes a local hydrophobic cluster which allosterically exposes the 306VQIVYK311 amyloid motif; identify a suppressor mutation that destabilizes S320F-based hydrophobic clustering reversing the phenotype in vitro and in cells; and computationally engineer spontaneously aggregating tau sequences through optimizing nonpolar clusters surrounding the S320 position. We uncover a mechanism for regulating tau aggregation which balances local nonpolar contacts with long-range interactions that sequester amyloid motifs. Understanding this process may permit control of tau aggregation into structural polymorphs to aid the design of reagents targeting disease-specific tau conformations.

Amyloid deposition of the microtubule-associated protein tau is a unifying theme in a multitude of neurodegenerative diseases. Disease-associated missense mutations in tau are associated with frontotemporal dementia (FTD) and enhance tau aggregation propensity. However, the molecular mechanism of how mutations in tau promote tau assembly into amyloids remains obscure. There is a need to understand how tau folds into pathogenic conformations to cause disease. Here we describe the structural mechanism for how an FTD-tau S320F mutation drives spontaneous aggregation. We use recombinant protein and synthetic peptide systems, computational modeling, cross-linking mass spectrometry, and cell models to investigate the mechanism of spontaneous aggregation of the S320F FTD-tau mutant. We discover that the S320F mutation drives the stabilization of a local hydrophobic cluster which allosterically exposes the 306VQIVYK311 amyloid motif. We identify a suppressor mutation that reverses the S320F aggregation phenotype through the reduction of S320F-based hydrophobic clustering in vitro and in cells. Finally, we use structure-based computational design to engineer rapidly aggregating tau sequences by optimizing nonpolar clusters in proximity to the S320 site revealing a new principle that governs the regulation of tau aggregation. We uncover a mechanism for regulating aggregation that balances transient nonpolar contacts within local protective structures or in longer-range interactions that sequester amyloid motifs. The introduction of a pathogenic mutation redistributes these transient interactions to drive spontaneous aggregation. We anticipate deeper knowledge of this process will permit control of tau aggregation into discrete structural polymorphs to aid design of reagents that can detect disease-specific tau conformations.

Protein assembly into β-sheet-rich amyloid structures is a general biophysical phenomenon that has significant biological consequences, most notable for their prominent association with neurodegenerative diseases, including Alzheimer's, Huntington's, or Parkinson's diseases. The assembly of amyloid structures is driven by short sequences called amyloid motifs. In many neurodegenerative diseases, intrinsically disordered proteins (IDPs) self-assemble through amyloid motifs, but these motifs are present in all proteins, including folded globular proteins. Importantly, mechanistic knowledge is lacking for how IDPs, which do not adopt a stable tertiary structure, mask these amyloidogenic motifs to mitigate or slow the formation of β-sheet-rich amyloid structures that cause disease. Our recent work has shown that local structural elements can modify the aggregation propensity of amyloid motifs in the intrinsically disordered microtubule-associated protein tau by adopting metastable β-hairpin-like structures that shield the amyloid motif, and disease-causing mutations change the conformation, thus increase aggregation propensity (Chen, Nat Commun 10:2493, 2019). Here we describe a protocol that correlates experimentally determined aggregation propensities for peptides measured by the Thioflavin T (ThT) fluorescence aggregation assay with their conformational ensembles derived from Groningen machine chemical simulations (GROMACS). Integration of experiment and simulation will help uncover structural rules behind changes in conformation that modulate protein aggregation. We anticipate that our general protocol will help identify key interactions in local structures that engage amyloid-forming motifs in IDPs which influence aggregation behavior.

The development of methyl transverse relaxation optimized spectroscopy has greatly facilitated the study of macromolecular assemblies by solution NMR spectroscopy. However, limited sample solubility and stability has hindered application of this technique to ongoing studies of complexes formed on membranes by the neuronal SNAREs that mediate neurotransmitter release and synaptotagmin-1, the Ca2+ sensor that triggers release. Since the 1H NMR signal of a tBu group attached to a large protein or complex can be observed with high sensitivity if the group retains high mobility, we have explored the use of this strategy to analyze presynaptic complexes involved in neurotransmitter release. For this purpose, we attached tBu groups at single cysteines of fragments of synaptotagmin-1, complexin-1 and the neuronal SNAREs by reaction with 5-(tert-butyldisulfaneyl)-2-nitrobenzoic acid (BDSNB), tBu iodoacetamide or tBu acrylate. The tBu resonances of the tagged proteins were generally sharp and intense, although tBu groups attached with BDSNB had a tendency to exhibit somewhat broader resonances that likely result because of the shorter linkage between the tBu and the tagged cysteine. Incorporation of the tagged proteins into complexes on nanodiscs led to severe broadening of the tBu resonances in some cases. However, sharp tBu resonances could readily be observed for some complexes of more than 200 kDa at low micromolar concentrations. Our results show that tagging of proteins with tBu groups provides a powerful approach to study large biomolecular assemblies of limited stability and/or solubility that may be applicable even at nanomolar concentrations., We thank Ad Bax for the suggestion of exploring the use tBu groups as probes for structural studies of the neurotransmitter release machinery, and Ad Bax, Lewis Kay and Charampalos Kalodimos for fruitful discussions on this subject. The Agilent DD2 console of the 800 MHz spectromenter used for the research presented here was purchased with a shared instrumentation grant from the NIH (S10OD018027 to JR). Rashmi Voleti was supported by a fellowship from the Howard Hughes Medical Institute. The preparation of BDSNB was performed at the NANBIOSIS –CIBER BBN Peptide Synthesis Unit (U3). This work was supported by grant I-1304 from the Welch Foundation (to JR) and by NIH Research Project Award R35 NS097333 (to JR).

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., The Hsp70/Hsp40 system plays an important role in maintaining cellular proteostasis but so far it is not well understood how Hsp70 proteins are recruited to specific Hsp40 co-chaperones. Here, the authors combine biochemical and biophysical approaches to characterise the oligomeric mammalian Hsp40 DnaJB8. They identify an intra-oligomer DnaJB8 interaction between the N-terminal J-Domain and the C-terminal domain that occludes the J-Domain surface that binds Hsp70 and propose a model for DnaJB8-Hsp70 recruitment.

In light of the high affinity of Cu2+ for Alzheimer's Aβ1–42 and its ability to subsequently catalyze the formation of radicals, we examine the effects of Cu2+ binding, Aβ oxidation, and an acidic environment on the conformational dynamics of the smallest Aβ1–42 oligomer, the Aβ1–42 dimer. Transition networks calculated from Hamiltonian replica exchange molecular dynamics (H-REMD) simulations reveal that the decreased pH considerably increased the β-sheet content, whereas Cu2+ binding increased the exposed hydrophobic surface area, both of which can contribute to an increased oligomerization propensity and toxicity.

Multiplication-stimulating activity (MSA) produced by Buffalo rat liver cells (BRL-3A) in culture is related to the somatomedin family of growth regulatory polypeptides. MSA will stimulate glucose transport and DNA synthesis in normal chicken embryo fibroblasts (CEF) at concentrations of 10-200 ng/ml. MSA found in BRL-3A-conditioned medium, like the somatomedins in serum, does not exist as the free hormone but is bound to a specific high molecular weight carrier protein. In this report we demonstrate that purified MSA carrier protein (MCP) inhibits the biological activity of MSA on CEF as measured by the stimulation of glucose transport and DNA synthesis. In addition, purified MCP competitively inhibits the binding of 125I-labeled MSA to these cells. In control experiments in which insulin was used as the mitogenic agent, MCP had no effect on these biological responses. These results indicate that the inhibitory effect of MCP is the result of specific interaction with MSA and support the hypothesis that cells may be unresponsive to somatomedins bound to their serum carrier proteins.

We report that pyridinium ions (HPyr+) accelerate the conversion of [Tp*MoIVOCl(OPMe3)] (1) to [Tp*MoIVOCl(NCCH3)] (2) by 103-fold, affording 2 in near-quantitative yield; Tp* = hydrotris(3,5-dimethyl-1-pyrazolyl)borate. This novel reactivity and the mechanism of this reaction were investigated in detail. The formation of 2 followed pseudo-first-order kinetics, with the observed pseudo-first-order rate constant (k obs) linearly correlated with [HPyr+]. An Eyring plot revealed that this HPyr+-facilitated reaction has a small positive value of ∆S ‡ indicative of a dissociative interchange (Id) mechanism, different from the slower associative interchange (Ia) mechanism in the absence of HPyr+ marked with a negative ∆S ‡. Interestingly, log(k obs) was found to be linearly correlated to the acidity of substituted pyridinium ions. This novel reactivity is further investigated using combined DFT and ab initio coupled cluster methods. Different reaction pathways, including Id, Ia, and possible alternative routes in the absence or presence of HPyr+, were considered, and enthalpy and free energies were calculated for each pathway. Our computational results further underscored that the Id route is energetically favored in the presence of HPyr+, in contrast with the preferred Ia–NNO pathway in the absence of HPyr+. Our computational results also revealed molecular-level details for the HPyr+-facilitated Id route. Specifically, HPyr+ initially becomes hydrogen-bonded to the oxygen atom of the Mo(IV)–OPMe3 moiety, which lowers the activation barrier for the Mo–OPMe3 bond cleavage in a rate-limiting step to dissociate the OPMe3 product. The implications of our results were discussed in the context of molybdoenzymes, particularly the reductive half-reaction of sulfite oxidase.