Understanding molecular features of aggregation-resistant tau conformer using oxidized monomer.

BACKGROUND: Aggregation of tau into paired helical filament (PHF) is a hallmark of Alzheimer's disease (AD), and Cys-mediated disulfide bond formation plays a vital role in tau fibrillation. While intermolecular disulfide bond between Cys residues in microtubule-binding repeat (MTBR) region facilitates tau aggregation, intramolecular disulfide bond attenuates the same, though the molecular basis for such phenomenon remains obscure. Thus intramolecular disulfide-bonded tau monomer might be an excellent model to understand the unique features of aggregation-resistant tau conformer. METHODS: We synthesized the Cys cross-linked tau40 monomer by oxidation and characterized the altered conformational dynamics in the molecule by Hydrogen-deuterium exchange, limited proteolysis and fluorescence quenching. RESULTS: Deuterium exchange study showed that rigidity was imparted in the core PHF region of oxidized tau40 in MTBR segment, consisting of the fundamental PHF6 motif. Conformational rigidity was prominent in C-terminal tail region also. Limited proteolysis supported reduced accessibility of MTBR region in the molecule. CONCLUSIONS: PHF formation of oxidized tau40 might be attenuated either by induction of intramolecular H-bonding between the regions of high ß-structure propensity in second and third MTBR (R2, R3), thus preventing intermolecular interaction between them, or by imparted rigidity in R2-R3, preventing the formation of extended ß-structure preceding fibrillation. Data indicated plausible effect of conformational adaptation on the nucleation process of oxidized tau40 assembly. GENERAL SIGNIFICANCE: Our findings unravel the essential molecular features of aggregation-resistant tau conformer. Therapeutics stabilizing such conformers in vivo might be of high benefit in arresting tau assembly during AD and other tauopathies.

Conformational heterogeneity of tau: Implication on intrinsic disorder, acid stability and fibrillation in Alzheimer's disease.

The self-assembly of intrinsically disordered protein tau into paired helical filament forms one of the hallmarks of Alzheimer's disease. However, the facets of innately disordered structure of tau and its conversion to a ß-sheet-rich fibril during several tauopathies are poorly understood. Here, we provide a direct insight into the ensemble of highly heterogeneous conformational families of tau at physiological pH, by nano-electrospray mass spectrometry coupled with ion mobility. The average collision cross section of the most unfolded conformer was higher by >2 fold than that of the most folded one. Acidic pH largely induced unfolding in tau, obliterating the compact conformers completely. The highly unfolded conformers were the key species bestowing the unusual solubility to tau at low pH, with limited contribution from intramolecular long-range interfaces giving rise to ordered conformers. Contrarily, alkaline pH shifted tau towards folded conformations due to charge neutralization, keeping the overall random coil architecture intact. Intriguingly, the heparin-induced in vitro aggregation propensity of the protein attenuated at both acidic and alkaline pH, illustrating the significance of altered conformations in pathological functions of tau. Our observations at low pH indicate that a reorganization of the intricate network of momentary long-range contacts in tau might have implication in its aggregation pathology. Disease-modifying therapies for Alzheimer's disease targeting either to disrupt the essential fibril-forming interaction at third microtubule-binding repeat of tau or to perturb specific binding interaction of tau with endogenous polyanionic species might be of high benefit.

Effect of altered solution conditions on tau conformational dynamics: Plausible implication on order propensity and aggregation.

Intrinsically disordered protein tau plays a central role in maintaining neuronal network by stabilizing microtubules in axon. Tau reportedly possesses random coil architecture, which is largely inert to alteration in solution conditions. However, the presence of transient compact conformers and residual structure has been evident from previous reports. Also, during Alzheimer's disease, misfolded tau detaches from microtubule and forms ordered filaments, which is the hallmark of the disease. Despite its fundamental role in neuronal physiology and in pathological cascade of several fatal neurodegenerative diseases, tau conformational dynamics remains poorly understood. In the present study, we have explored the effect of ionic strength, temperature and solvent polarity on tau40 conformational preferences using ion mobility mass spectrometry. Investigation of collision cross section revealed that while low ionic strength, elevated temperature and reduced solvent polarity mostly induced partial collapse in tau40 conformers, higher ionic strength led to an expansion of the molecule. Limited proteolysis identified segments of tau40 projection domain and proline-rich region having high order propensity and a C-terminal region having vulnerability for further expansion at altered solution conditions. The high susceptibility for disorder-to-order transition in the above region of the protein might have crucial implication on its role as microtubule spacers, and in cellular signaling cascade. The conformational adaptation of tau40 did not enhance the heparin-induced aggregation proclivity of the protein. Nevertheless, the observed correlation of electrostatic interaction with fibrillation propensity of tau40 might indicate plausible link between hyperphosphorylation at diseased state with tau conformation and self-assembly.