Structural Impact of Heparin Binding to Full-Length Tau As Studied by NMR Spectroscopy

Abstract

The neuronal Tau protein is involved in stabilizing microtubules but is also the major component of the paired helical filaments (PHFs), the intracellular aggregates that characterize Alzheimer's disease (AD) in neurons. In vitro, Tau can be induced to form AD-like aggregates by adding polyanions such as heparin. While previous studies have identified the microtubule binding repeats (MTBRs) as the major player in Tau aggregation, the fact that the full-length protein does not aggregate by itself indicates the presence of inhibitory factors. Charge and conformational changes are of uttermost importance near the second (R2) and third (R3) MTBR that are thought to be involved directly in the nucleation of the aggregation. Recently, the positively charged regions flanking the MTBR were proposed to inhibit PHF assembly, where hyperphosphorylation neutralizes these basic inhibitory domains, enabling Tau-Tau interactions. Here we present results of an NMR study on the interaction between intact full-length Tau and small heparin fragments of well-defined size, under conditions where no aggregation occurs. Our findings reveal (i) micromolar affinity of heparin to residues in R2 and R3, (ii) two zones of strong interaction within the positively charged inhibitory regions flanking the MTBR, and (iii) another interaction site upstream of the two inserts encoded by exons 2 and 3. Three-dimensional heteronuclear NMR experiments demonstrate that the interaction with heparin induces beta-strand structure in several regions of Tau that might act as nucleation sites for its aggregation but indicate as well alpha-helical structure in regions outside the core of PHF. In the PHF, the residues outside of the core maintain sufficient mobility for NMR detection and recover their unbound chemical shift values after an overnight incubation at 37 degrees C with heparin. Heparin thus becomes integrated into the rigid core region of the PHF, probably providing the charge compensation for the lysine-rich stretches that form upon the in-register, parallel stacking of the repeat regions.

Pharmacology of Heparin and Related Drugs

“…Heparin interacts with t at several sites on the protein, inducing changes in secondary structure and becoming incorporated into paired helical filaments (Sibille et al, 2006). The propagation of t aggregates through neural networks, involving release of t into the extracellular space and uptake by adjacent cells, has been recognized as a major mechanism for the spread of neurofibrillary pathology (Medina and Avila, 2014), which may involve HS as a cell surface receptor (Holmes et al, 2013).…”

Understanding the Kinetic Roles of the Inducer Heparin and of Rod-like Protofibrils during Amyloid Fibril Formation by Tau Protein

“…5c). Because the binding of heparin to Tau is tight, with a dissociation constant in the sub-micromolar region (44,45), the hyperbolic dependence of the extent of fibril formation on heparin concentration (Fig. 5c) can be used to estimate reliably the stoichiometry of protein bound to heparin in the ratelimiting steps of aggregation; one heparin molecule appears to bind only 2-3 protein molecules.…”

“…Heparin (H) brings two positively charged monomeric protein (P) molecules together, thus enabling them to interact and undergo the conformational change necessary to form the aggregation nucleus (PHP). Indeed, heparin has been shown to induce conformational changes in Tau (44,46), and a role for heparin in regulating the conformation of Tau has been proposed earlier (30). It has also been postulated that heparin functions by binding sites of residual ␤ structure and facilitating ␤-strand interactions across different Tau molecules (38).…”

“…The explanation based on the existence of a low affinity binding site for heparin, binding to which at high heparin concentrations would reduce the concentration of the aggregation-competent PHP molecules, is plausible (31), especially as it is known that Tau has multiple heparin binding sites (44), but it requires a more complex model than the minimal model shown in Fig. 8.…”

The Microtubule-Dissociating Tau in Neurological Disorders

“…Induction of tau fibrillization in cells remain unsatisfactory, this is a limiting factor since NFD cannot be totally reproduced in vitro (Sibille N et al, 2006). The development of in vivo models has provided an important tool to precise sequence of molecular events leading to tau aggregation.…”