Two Tau binding sites on tubulin revealed by thiol-disulfide exchanges

Abstract

Tau is a Microtubule-associated protein that induces and stabilizes the formation of the Microtubule cytoskeleton and plays an important role in neurodegenerative diseases. The Microtubules binding region of Tau has been determined for a long time but where and how Tau binds to its partner still remain a topic of debate. We used Site Directed Spin Labeling combined with EPR spectroscopy to monitor Tau upon binding to either Taxol-stabilized MTs or to αβ-tubulin when Tau is directly used as an inducer of MTs formation. Using maleimide-functionalized labels grafted on the two natural cysteine residues of Tau, we found in both cases that Tau remains highly flexible in these regions confirming the fuzziness of Tau:MTs complexes. More interestingly, using labels linked by a disulfide bridge, we evidenced for the first time thiol disulfide exchanges between αβ-tubulin or MTs and Tau. Additionally, Tau fragments having the two natural cysteines or variants containing only one of them were used to determine the role of each cysteine individually. The difference observed in the label release kinetics between preformed MTs or Tau-induced MTs, associated to a comparison of structural data, led us to propose two putative binding sites of Tau on αβ-tubulin.

The mechanism of Hsp90-induced oligomerizaton of Tau

“…This is a typical way for Hsp90 to bind its client proteins (50,51). In addition, Tau typically retains a high degree of conformational dynamics, at least for protein segments, when in complex with various inter action partners including heparin, tubulin, and MTs and in Tau fibrils (9,26,38,52,53). Binding to Hsp90 induces a conformational opening of paper clip-Tau (7,8), leading to exposure of Tau-RD.…”

Tau isoform–specific stabilization of intermediate states during microtubule assembly and disassembly

“…Multiple tau-binding sites upon MTs have been reported, including sites along the outer MT surface (37)(38)(39)(40), at the longitudinal interface between heterodimers (41), and within the MT lumen (interior) (42,43). Importantly, kinetic analyses in vitro (44) and in cells (45) indicate that tau has at least two modes of binding to MTs, one that is transient and readily exchangeable with tau in solution and another that is relatively nonexchangeable.…”

Tau repeat regions contain conserved histidine residues that modulate microtubule-binding in response to changes in pH

“…Although a high-definition model of native MT-bound tau is still not available, recent cryo-EM data of MTbound constructs of human tau MT-binding repeat R2 show a close interaction of His299 with the tubulin interdimer interface (28). This overall arrangement of tau histidine residues near tubulintubulin interfaces is also supported by NMR and more recent electron paramagnetic resonance spectroscopy data (30,39).…”