Nonsaturable binding indicates clustering of Tau on the microtubule surface in a paired helical filament-like conformation

Tau protein modulates microtubule dynamics and forms insoluble aggregates i n Alzheimer's disease. Because there is a discrepancy between reported affi nities of Tau to microtubules, we determined the interaction over a wide co ncentration range using a sensitive enzyme -linked immunosorbent assay. We found that the interaction is biphasic and not monophasic as assumed earlie r. The first binding phase is typical for identical and noninteracting bind ing sites, with dissociation constants around 0.1 mu M and stoichiometries around 0.2 Tau/tubulin dimer. Surprisingly, the second phase is nonsaturabl e and shows a nearly linear increase in bound Tau versus free Tau for free Tau concentrations higher than 2 mu M. The slope is proportional to the mic rotubule concentration. From this we define an overloading parameter with v alues around 50 mu M. The influence of Tau isoform, phosphorylation, and di merization on both phases was investigated. Remarkably the overloading of T au on microtubules leads to a thioflavin S fluorescence increase reminiscen t of that seen with Tau aggregated into Alzheimer paired helical filaments. Because polyanions stimulate Tau aggregation and because the C-terminal do main of tubulin is polyanionic, we suggest that an early conformational cha nge in Tau leading to paired helical filament aggregation occurs right on t he microtubule surface.