Investigation of tau-tubulin interaction: The role of intrinsic disorder in tau's function and dysfunctio

Tau is a microtubule-associated protein which functions to stabilize microtubules in axons of neurons. Its implication in a number of neurodegenerative diseases including Alzheimer's disease and frontotemporal dementias, collectively known as tauopathies, makes it a key target for drug discovery and therapies treating neurodegenerative diseases. Both gain-of-toxicity and loss-of-normal-function are thought to be relevant to tau pathology. Understanding the detailed molecular mechanism of tau function would be crucial in developing treatments targeting tau, as well as contributing to our understanding of how tau regulates microtubule dynamics. Although there it has been heavily studied, detailed mechanisms for tau's native function remain to be elucidated. To illustrate, prior studies of tau function have primarily focused on its interaction with microtubules, while the interaction between tau and tubulin has been largely overlooked.;In this dissertation, I aim to achieve a molecular description of tau-tubulin interaction in order to gain insight into how this contributes to tau native function. First, I confirmed that tau binds to tubulin with a high affinity and found that altered binding affinities between tau and tubulin may play a role in dysfunction caused by disease-related mutations.;I then investigated structural features of tau-tubulin complexes. Using fluorescence correlation spectroscopy (FCS), I found that tau is able to bind multiple tubulin dimers even under conditions when the polymerization of microtubule is inhibited. The use of polymerization-inhibiting engineered proteins allowed me to demonstrate that tau is able to mediate both lateral and longitudinal interaction between tubulin dimers. Furthermore, I developed an assay based on acrylodan fluorescence screening to characterize conformational changes in tau upon binding to tubulin at single-residue resolution. My results revealed that a hierarchy amongst the binding repeats may exist for tubulin binding. A partial disorder-to-helical transition was also observed when tau binds to tubulin. I proposed a model to explain how tau interacts with soluble tubulin dimers, where short segments in binding repeats may serve as hot spots mediating direct interaction by a disorder-to-helical transition, while the other parts of tau stay disordered to provide the necessary flexibility of the chain for this interaction.;Finally, I correlate structural features with functional insights. I found that tau binds to tubulin to form `fuzzy complexes' with variable stoichiometry and the pseudo-repeat region following the microtubule-binding repeat plays major role in mediating this heterogeneity. Interestingly, I found strong correlation between the ability of tau to bind to multiple tubulin dimers and its effectiveness in polymerizing tubulin. Our findings provide another missing link in explaining tau's native function, as well as a good example to illustrate how an intrinsically disordered protein functions to regulate a highly dynamic system.