Complementary dimerization of microtubule-associated tau protein: Implications for microtubule bundling and tau-mediated pathogenesis

Abstract

Tau is an intrinsically unstructured microtubule (MT)-associated protein capable of binding to and organizing MTs into evenly spaced parallel assemblies known as "MT bundles." How tau achieves MT bundling is enigmatic because each tau molecule possesses only one MT-binding region. To dissect this complex behavior, we have used a surface forces apparatus to measure the interaction forces of the six CNS tau isoforms when bound to mica substrates in vitro. Two types of measurements were performed for each isoform: symmetric configuration experiments measured the interactions between two tau-coated mica surfaces, whereas "asymmetric" experiments examined tau-coated surfaces interacting with a smooth bare mica surface. Depending on the configuration (of which there were 12), the forces were weakly adhesive, strongly adhesive, or purely repulsive. The equilibrium spacing was determined mainly by the length of the tau projection domain, in contrast to the adhesion force/energy, which was determined by the number of repeats in the MT-binding region. Taken together, the data are incompatible with tau acting as a monomer; rather, they indicate that two tau molecules associate in an antiparallel configuration held together by an electrostatic "zipper" of complementary salt bridges composed of the N-terminal and central regions of each tau monomer, with the C-terminal MT-binding regions extending outward from each end of the dimeric backbone. This tau dimer determines the length and strength of the linker holding two MTs together and could be the fundamental structural unit of tau, underlying both its normal and pathological action.

Fig. 1.

A schematic of the six CNS tau isoforms, with their associated charge distributions. (A) The six isoforms, generated by alternative RNA splicing, differ by the presence of either three or four 18-aa-long imperfect repeats (red), separated from one another by a 13-to 14-aa interrepeat (orange), in the C-terminal half of the protein and the presence of zero, one, or two 29-aa inserts in the N-terminal half of the protein. (B) Charge-distribution plot (using 10-aa windows) shows that the N terminus is negatively charged and the C terminus is positively charged.

Fig. 2.

Force data for the 3R0N tau isoform. (A) Symmetric configuration. (B) Asymmetric configuration. For each, the force F is normalized by the radius of curvature of the mica surfaces R and plotted versus the surface separation distance D. Filled squares and “In” curve: forces measured between approaching surfaces; open circles and “Out” curve: forces measured between receding surfaces. There are two instabilities where surfaces spontaneously jump in or out: D_J is the jump-in separation, which gives the “range” of the interaction, and D_eq is the jump-out separation, which gives the “equilibrium” configuration and lowest (binding) energy, E_eq = F_ad/2πR.

Fig. 3.

Characteristic ranges of the binding interactions (given by D_J) and magnitudes of the adhesion energies (given by E_eq = F_ad/2πR) for the 12 configurations studied.

Fig. 4.

Cartoon model of bivalent tau dimer held together by an electrostatic zipper and interacting with two mica (model MT) surfaces.

Fig. 5.

Cartoon model of bivalent tau dimer associating with and bundling MTs. (A) The amino acid proposed to form the electrostatic zipper is emphasized. (B) Bivalent tau dimers bundling MTs is depicted. The electron micrograph of bundled MTs is from ref. [adapted by permission from Macmillan Publishers Ltd: Nature (2), copyright 1992].

Fig. 6.

The total interaction energy E decomposed into the fundamental steric, electrostatic (es), and mica–mica bridging forces for the symmetric (A) and asymmetric (B) cases. The tau molecules in bold are those involved in a bridging interaction.