Structural and biophysical characterization of the projection domain of neuronal tau protein

Nina Zemanova1, Rostislav Skrabana2,3, Maria Janubova3, Ondrej Cehlar2,3, Michal Novak2,3

1Faculty of Natural Sciences, Comenius University, Mlynska dolina, Ilkovicova 6, 842 15 Bratislava 4, Slovakia,

2Institute of Neuroimmunology, Dubravska cesta 9, 845 10 Bratislava, Slovakia

3Axon Neuroscience, Dvorakovo nabrezie 10, 811 02 Bratislava, Slovakia


Tau protein belongs to intrinsically disordered proteins (IDPs). IDPs do not have well defined secondary and tertiary structure. Under physiological conditions, IDP tau promotes tubulin monomers assembly, binds to axonal microtubules and stabilizes them. However, in a group of neurodegenerative diseases called tauopathies, tau protein aggregates and accumulates in the entire neuron forming insoluble fibrillary deposits – neurofibrillary tangles. The most prevalent tauopathy is Alzheimer’s disease, which today is one of the most serious health problems in the world.

It is little known about the structure of tau protein and its pathological transformations. Recent evidences suggest that N-terminal region of tau protein is critical for the stabilization and organization of certain types of axons. In this work we are focusing on structural and biophysical characterization of N-terminal projection domain of tau protein to bring some new insights about its conformational and dynamic features. Monoclonal antibody AX-N1, which recognizes an epitope in the first alternatively spliced N-terminal insert of tau was used as a molecular probe.

We have performed a crystallization study of AX-N1 antibody Fab fragment alone and in the complex with tau peptides. We have found crystallization conditions for all tested systems; crystals of Fab fragment alone diffracted to 3.8 Å on a laboratory X-ray source. For biophysical characterisation we used ELISA and surface plasmon resonance (SPR). With SPR we have determined binding rate constants of tau polypeptide 31-441 and tau protein 1-441 with AX-N1 Fab fragment. The results show that full-length tau protein isoform has higher association rate constant and lower dissociation rate constant then truncated tau. cELISA revealed that the AX-N1 affinity is enhanced in the presence of the second alternatively spliced N-terminal insert and in the absence of the second alternatively spliced repeat in the C-terminal part. Therefore, the three-repeat tau protein isoforms are more prone to bind the antibody then the four-repeat isoforms. Obtained results suggest different configuration of the first tau insert in the context of various tau protein isoforms.