Tau Interdomain Relationships: Re-examining the Role of the Proline Rich Region in Tau's Structure and Function in vitro
Tau is an intrinsically disordered (IDP), microtubule-associated (MAP) protein that has a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of tau-mediated microtubule polymerization are poorly understood. In particular, although significant effort has poured into interactions between the microtubule protofilament and one of tau’s regions, the microtubule-binding region (MTBR), the remaining tau domains and interactions with unincorporated soluble tubulin remain understudied. Here, we used single-molecule fluorescence to investigate the role of tau’s N-terminal domain (NTD) and proline-rich region (PRR) in regulating interactions of tau with soluble tubulin. We assayed both full-length tau isoforms and truncated variants for their ability to bind soluble tubulin and stimulate microtubule polymerization. We found that tau’s PRR is an independent tubulin-binding domain that has tubulin polymerization capacity. In contrast to the relatively weak interactions with tubulin mediated by sites distributed throughout tau’s MTBR, resulting in heterogeneous tau:tubulin complexes, the PRR bound tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. On the basis of these results, we propose that tau’s PRR can serve as a core tubulin-binding domain, whereas the MTBR enhances polymerization capacity by increasing the local tubulin concentration. Moreover, the NTD appears to negatively regulate tubulin-binding interactions of both of these domains. The findings of our study draw attention to a central role for the PRR in tau function and provide mechanistic insight into tau-mediated polymerization of tubulin. However, PRR binding was not observed in the recent cryo-EM structure of tau’s MTBR bound to the microtubule protofilament (Nogales, 2018). Using an environmentally sensitive fluorophore acrylodan, we demonstrate similarities between tau’s MTBR structure to soluble tubulin and the microtubule. Our preliminary work suggests PRR binding to tubulin is strongly mediated through the tubulin tails. In summary, this thesis provides mechanistic and structural insight into tau-mediated microtubule polymerization by (1) clarifying the role of tau’s PRR thereby refining polymerization models (2) demonstrating a conserved ensemble between the NTD and the PRR/MTBR regions of functional significance, and (3) outlining preliminary groundwork for contrasting tau-microtubule and tau-tubulin interactions.
McKibben, Kristen M, "Tau Interdomain Relationships: Re-examining the Role of the Proline Rich Region in Tau's Structure and Function in vitro" (2020). Dissertations available from ProQuest. AAI27958258.