Final Report Summary - HSP70-TAU NMR (Structural analysis of the conformational transitions of the K18 fragment of human tau driven by Hsp70 action) Aggregation of Tau is a central hallmark in the pathogenic cascade of Alzheimer’s disease. In physiological conditions this so-called neurotoxic protein adopts a disordered conformation but undergoes conformational transitions leading to its aggregation and amyloid fibril formation under neuronal stress1. ATP-driven molecular chaperones of the heat-shock protein (Hsp) family have been postulated to play a critical role inhibiting the gain-of-toxic function of these neurotoxic proteins2. However, little is known about the binding and activation mechanism of neurotoxic proteins, and of disordered proteins in general, induced by Hsps. Given the dynamic and unstable nature of these interactions and activation cycles, Nuclear Magnetic Resonance spectroscopy is ideally suited to obtain insight into the nature of these interactions. In this project we aimed to characterize the binding of Tau to the different conformations of Hsp70, and on the basis of observable transferred NOEs3 on small fragments that would cover the binding region, we planned to provide a detailed structural characterization of an ensemble of Tau conformers bound to each allosteric form of Hsp70. We also proposed to study the role of Hsp40 in this complex formation in order to understand the mechanism of substrate transfer between Hsp40-Hsp70 chaperones2. All this information would be used to understand the structural basis of the effect of methylthionine derivatives on this complex, since they were found to specifically inhibit Tau aggregation as well as Hsp70 ATPase activity4,5. In this project we have obtained - by means of novel artful NMR experimental designs, complemented with extensive biochemical and biophysical characterization – detailed information on the binding mechanism of the aggregation-prone fragment of Tau to the two different allosteric conformations of Hsp70. Furthermore we have determined the different regions of Tau that bind to Hsp70 and Hsp40, providing valuable insights into the Tau-Hsp40-Hsp70 ternary complex. We also investigated mechanistic details of the structural recognition of Tau by Hsp90 chaperone. We have found that different regions of Tau are specifically recognized by the different chaperones. Besides, Tau is recognized by specific regions of the chaperones, which are different from those used for the binding of folded substrates. This interplay between Tau (a long disordered amyloidogenic protein) and the different chaperones might regulate Tau’s fate inside the cell. These findings for the first time shed light onto the still unsolved conformational changes in a client protein motivated by the chaperone mode of action. Moreover, they provide invaluable information on the structural recognition of misfolded proteins by Hsps. Detailed knowledge on the structure and dynamics of Hsps-client proteins is of paramount importance to fully understand how multiple roles of Hsps are regulated. Besides, it is a first step to propose the use of compounds that specifically manipulate these dynamic complexes, in order to the design drugs for the treatment of amyloid disorders6. Because amyloidogenic neurodegenerative diseases are incurable conditions with high prevalence in modern society (more than 7 million people suffer from amyloidogenic proteinopathies in the EU), the enormous socio-economic impact of such discoveries can be clearly envisioned. The total yearly cost of these type of illnesses in Europe is more than 160 million Euro, and therefore there is a very high interest in the scientific and pharmaceutical worlds to develop new specific potent drugs that could cure neurodegenerative diseases. One of the most important first steps towards this end is to fully understand the pathogenic mechanisms that trigger the disease and the role of the different regulators in this pathogenic cascade. This project was designed along these lines and has successfully provided invaluable insights into these mechanisms. 1. Chiti F. and Dobson C.M. (2006) Ann. Rev. Biochem. 75 : 333-66.2. Hartl F.U. Bracher A. and Hayer-Hartl M. (2011) Nature 475 (7356): 324-32.3. Carlomagno T. (2005) Annu. Rev. Biophys. Biomol. Struct. 34 : 245-66.4. Wischik C.M. et al. (1996) Proc. Natl. Acad. Sci. USA 93 (20): 11213-8.5. Akoury E. et al. (2013) Angew. Chem. Int. Ed. Engl. 52 (12) : 3511-5.6. Johnson S.M. et al. (2012) J. Mol. Biol. 421: 185-203.
