The structure, dynamic behavior, and spatial organization of microtubules are regulated by microtubule-associated proteins. An important microtubule-associated protein is the protein Tau, because its microtubule interaction is impaired in the course of Alzheimer's disease and several other neurodegenerative diseases. Here, we show that Tau binds to microtubules by using small groups of evolutionary conserved residues. The binding sites are formed by residues that are essential for the pathological aggregation of Tau, suggesting competition between physiological interaction and pathogenic misfolding. Tau residues in between the microtubulebinding sites remain flexible when Tau is bound to microtubules in agreement with a highly dynamic nature of the Tau-microtubule interaction. By binding at the interface between tubulin heterodimers, Tau uses a conserved mechanism of microtubule polymerization and, thus, regulation of axonal stability and cell morphology.

A network of molecular chaperones is known to bind proteins (“clients”) and balance their folding, function and turnover. However, it is often not clear which chaperones are critical for selective recognition of individual clients. It is also not clear why these key chaperones might fail in protein aggregation diseases. In this study, we utilized human microtubule-associated protein tau (MAPT or tau) as a model client to survey interactions between ~30 purified chaperones and ~20 disease-associated tau variants (~600 combinations). From this large-scale analysis, we identified human DnaJA2 as an unexpected, but potent, inhibitor of tau aggregation. DnaJA2 levels were correlated with tau pathology in human brains, supporting the idea that it is an important regulator of tau homeostasis. Of significance, we found that some disease-associated tau variants were relatively immune to interactions with chaperones, suggesting a model in which avoiding physical recognition by chaperone networks may contribute to disease. Impact Statement Large-scale screening of chaperone interactions with tau and its variants identified DnaJA2 as a key protective factor in tauopathy.

Microtubules are regulated by microtubule-associated proteins. However, little is known about the structure of microtubule-associated proteins in complex with microtubules. Herein we show that the microtubule-associated protein Tau, which is intrinsically disordered in solution, locally folds into a stable structure upon binding to microtubules. While Tau is highly flexible in solution and adopts a β-sheet structure in amyloid fibrils, in complex with microtubules the conserved hexapeptides at the beginning of the Tau repeats two and three convert into a hairpin conformation. Thus, binding to microtubules stabilizes a unique conformation in Tau.

Phosphorylation of the microtubule-associated protein Tau influences the assembly and stabilization of microtubules and is deregulated in several neurodegenerative diseases. The high flexibility of Tau, however, has prevented an atomic-level description of its phosphorylation-induced structural changes. Employing an extensive set of distance and orientational restraints together with a novel ensemble calculation approach, we determined conformational ensembles of Tau fragments in the non-phosphorylated state and, when phosphorylated at T231/S235 or T231/S235/S237/S238, four important sites of phosphorylation in Alzheimer disease. Comparison of the molecular ensembles showed that phosphorylation of the regulatory T231 does not perturb the backbone conformation of the proximal microtubule-binding (225)KVAVVR(230) motif. Instead, phosphorylated T231 selectively engages in a salt bridge with R230 that can compete with the formation of intermolecular salt bridges to tubulin. Our study provides an ensemble description which will be useful for the analysis of conformational transitions in Tau and other intrinsically disordered proteins.

Cellular condensation of intrinsically disordered proteins (IDPs) through liquid-liquid phase separation (LLPS) allows dynamic compartmentalization and regulation of biological processes. The IDP tau, which promotes the assembly of microtubules and is hyperphosphorylated in Alzheimer's disease, undergoes LLPS in solution and on the surface of microtubules. Little is known, however, about the influence of tau phosphorylation on its ability to nucleate microtubule bundles in conditions of tau LLPS. We here show that unmodified tau as well as tau phosphorylated at disease-associated epitopes condense into liquid-like droplets. Although tubulin partitioned into and reached high concentrations inside all tau droplets, it was unable to grow into microtubules form the inside of droplets formed by tau phosphorylated at the AT180 epitope (T231/S235). In contrast, neither phosphorylation of tau in the repeat domain nor at its tyrosine residues inhibited the assembly of tubulin from tau droplets. Because LLPS of IDPs has been shown to promote different types of cytoskeletal assembly, our study suggests that IDP phosphorylation might be a broadly used mechanism for the modulation of condensate-mediated cytoskeletal assembly.

Tau protein plays an important role in neuronal physiology and Alzheimer's neurodegeneration. Its abilities to aggregate abnormally, to bind to microtubules (MTs), and to promote MT assembly are all influenced by phosphorylation. Phosphorylation of serine residues in the KXGS motifs of Tau's repeat domain, crucial for MT interactions and aggregation, is facilitated most efficiently by microtubule-associated protein/microtubule affinity-regulating kinases (MARKs). Here we applied high-resolution nuclear magnetic resonance analysis to study the kinetics of phosphorylation of Tau by MARK2 and its impact on the structure and microtubule binding of Tau. We demonstrate that MARK2 binds to the N-terminal tail of Tau and selectively phosphorylates three major and five minor serine residues in the repeat domain and C-terminal tail. Structural changes induced by phosphorylation of Tau by MARK2 are highly localized in the proximity of the phosphorylation site and do not affect the global conformation, in contrast to phosphorylation in the proline-rich region. Furthermore, single-residue analysis of binding of Tau to MTs provides support for a model in which Tau's hot spots of MT interaction bind independently of each other and are differentially affected by phosphorylation.

The microtubule-associated protein Tau promotes the polymerization of tubulin and modulates the function of microtubules. As a consequence of the dynamic nature of the Tau-tubulin interaction, the structural basis of this complex has remained largely elusive. By using NMR methods optimized for ligand-receptor interactions in combination with site-directed mutagenesis we demonstrate that the flanking domain downstream of the four microtubule-binding repeats of Tau binds competitively to a site on the α-tubulin surface. The binding process is complex, involves partial coupling of different interacting regions, and is modulated by phosphorylation at Y394 and S396. This study strengthens the hypothesis of an intimate relationship between Tau phosphorylation and tubulin binding and highlights the power of the INPHARMA NMR method to characterize the interaction of peptides derived from intrinsically disordered proteins with their molecular partners.

Microtubule-associated proteins regulate microtubule dynamics, bundle actin filaments, and cross-link actin filaments with microtubules. In addition, aberrant interaction of the microtubule-associated protein Tau with filamentous actin is connected to synaptic impairment in Alzheimer’s disease. Here we provide insight into the nature of interaction between Tau and actin filaments. We show that Tau uses several short helical segments to bind in a dynamic, multivalent process to the hydrophobic pocket between subdomains 1 and 3 of actin. Although a single Tau helix is sufficient to bind to filamentous actin, at least two, flexibly linked helices are required for actin bundling. In agreement with a structural model of Tau repeat sequences in complex with actin filaments, phosphorylation at serine 262 attenuates binding of Tau to filamentous actin. Taken together the data demonstrate that bundling of filamentous actin and cross-linking of the cellular cytoskeleton depend on the metamorphic and multivalent nature of microtubule-associated proteins.