Kadavath, Harindranath

English

Tau is an intrinsically disordered, microtubule associated protein and is responsible for the promotion of microtubule (MT) formation and stabilization. The human Tau protein has six different isoforms, which are expressed in neurons of the central nervous system. A precise understanding of the molecular mechanism involved in the Tau-MT interaction is highly relevant to the study of neurodegenerative states as irregularities in the function of these proteins may lead to neuronal loss and cell death. For instance, the hyperphosphorylation of Tau leads to the depolymerization of MTs and formation of neurofibrillary tangles of Tau which is one of the hallmarks in neurodegenerative diseases. 
Although the function of Tau and MTs had been studied previously, it was still unclear how Tau binds to microtubules and high resolution information allowing the structural characterization of microtubule-bound Tau was missing. The aim of the thesis was to perform an in-depth study of the Tau-MT interaction and to determine the MT-bound conformation of the Tau protein. Furthermore, the identification of the binding sites of Tau on MTs will provide a three-dimensional view of the MT-bound Tau structure. 
Among the available techniques to derive structural information, NMR is the only method that allows structural description in near-physiological conditions and at atomic resolution. A divide-and-conquer approach in combination with exchange-transferred NMR methods were employed to overcome the size limitations in NMR and to establish the three-dimensional structure of MT-bound Tau.
This thesis covers the detailed NMR characterization of different Tau isoforms, mutants and shorter Tau constructs, thereby identifying the potential binding hot spots of Tau involved in MT interaction. This allowed the structure determination of highly independent and dynamic binding domains of MT-bound Tau using a ‘divide and conquer’ strategy and tr-NOE method. Sufficient experimental evidences obtained using NMR and other biophysical methods proved that the shorter Tau fragments used for structure determination can function similar to the full-length Tau protein. The influence of site-specific mutations and phosphorylation of Tau and Tau fragments is correlated with the structure that we identified. The obtained three-dimensional structure thus provides insights into the structure-function relationship of MT-bound Tau. Competition experiments using Tau fragments and MT targeting drugs in combination with tr-NOE, STD and Inpharma experiments revealed that the Tau binding domains compete against each other for binding to a single binding site on tubulin near the binding site of vinblastine. The results are supported by chemical cross linking coupled with mass spectrometry. Together with this information, it is possible to suggest a three-dimensional model of microtubule-bound Tau which is relevant to neurodegenerative diseases

Georg-August-University Göttingen

NMR Spectroscopy of the Tau-Microtubule Interaction

Kadavath, H.

MPG.PuRe