The scientific history of tau protein is closely linked to the discovery of microtubule self-assembly in the early 1970s. Previously, tubulin was known as a major "colchicine-binding protein" in the brain (BorisyandTaylor 1967),butthe conditions for assembling this protein into microtubules remained elusive until they were found by Weisenberg (1972) and others. This finding paved the way for identifying the central roles of microtubules for cell division, cell shape, and intracellular transport. An early key observation was that microtubule assembly was facilitated by microtubule-associated proteins, one of which was tau protein, isolated from brain by Kirschner's group (Weingarten et al. 1975).
The protein has unusual properties in that it is heat-stable and acid-stable, i.e., it is highly soluble so that it does not precipitate during boiling and treatment with acids. Its spectral properties are characteristic of a "random coil" protein (Cleveland et al. 1977). Imaging of tau by electron microscopy gave ambiguous results due to its low contrast (Zingsheim et al. 1979). Special preparation techniques such as quick-freeze deep-etching revealed a microtubule-bound "assembly domain" (so called because this domain promotes the assembly of microtubules) and a "projection domain" that protrudes away from the microtubule wall (Hirokawa et al. 1988). Image reconstructions from unstained microtubules decorated with tau molecules confirmed the largely disordered nature of tau on the surface (Santarella et al. 2004).
The cloning of tau from mouse and human (Lee et al.1987; Goedert et al. 1989b) revealed the sequence and domain composition (Fig. 1). Tau is unusually rich in polar
1 Max-Planck-Unit for Structural Molecular Biology, c/o DESY, Notkestrasse 85,22607 Hamburg, Germany
Fig. 1. Diagram oftau domains. The bar illustrates Tau441, the longest isoform in human CNS. Inserts N1, N2, and R2 can be alternatively spliced, giving rise to six isoforms. The N-terminal domain up to ~G120 has an acidic character; the other domains are basic. The left half (residues 1 to ~ 200) represents the "projection domain," the right half the "microtubule assembly domain". The 3 or 4 repeats R1-R4 comprise the core of the microtubule-binding domain as well as the core of the paired helical filaments (PHF). Two hexapeptide motifs at the beginning of R2 and R3 promote PHF aggregation by inducing ^-structure. AK280 and P301L are two mutants from FTDP-17 cases that strongly enhance the rate of PHF aggregation by increasing the propensity for p-structure
Fig. 1. Diagram oftau domains. The bar illustrates Tau441, the longest isoform in human CNS. Inserts N1, N2, and R2 can be alternatively spliced, giving rise to six isoforms. The N-terminal domain up to ~G120 has an acidic character; the other domains are basic. The left half (residues 1 to ~ 200) represents the "projection domain," the right half the "microtubule assembly domain". The 3 or 4 repeats R1-R4 comprise the core of the microtubule-binding domain as well as the core of the paired helical filaments (PHF). Two hexapeptide motifs at the beginning of R2 and R3 promote PHF aggregation by inducing ^-structure. AK280 and P301L are two mutants from FTDP-17 cases that strongly enhance the rate of PHF aggregation by increasing the propensity for p-structure and charged amino acids and has abasic character (except for the initial ~ 120 residues, where negative charges dominate). This explains the high solubility and the unfolded nature of the protein; however, it makes the aggregation of the protein in AD even more enigmatic. In particular, at first glance the sequence contains no elements that appear particularly amyloidogenic, such as exposed stretches of hydrophobic residues (as in the Ap peptide of AD) or glutamines (as in huntingtin). Tau occurs in a number of isoforms, typically six in the human CNS, which arise from alternative mRNA splicing of exons 2, 3, and 10 and generate isoforms containing 352-441 amino acid residues. The N-terminal projection domain and C-terminal assembly domain can be separated by chymotryptic cleavage behind Y197 (Steiner et al. 1990); the C-terminal tail can be removed by caspase 3 behind D421 (Gamblin et al. 2003). The most conspicuous feature is the repeat domain within the C-terminal half (Q244-N368), containing three or four semi-conserved sequences of 31 or 32 residues: R1 = Q244-K274, R2 = V275-S305, R3 = V306-Q336, R4 = V337-N368. R2 is encoded by exon 10 and maybe absent. The resulting isoforms can be designated as 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, 2N4R, depending on the number of N-terminal inserts and repeats. In peripheral nerves, additional isoforms can occur by inclusion of more exons encoding ~ 300 further residues, generating "big tau" (Couchie et al. 1992). The general domain structure of tau is similar to other microtubule-associated proteins, such as the neuronal MAP2 or the ubiquitous MAP4, which, however, contain a much larger projection domain (Lewis et al. 1988). Its size determines the spacing between microtubules in cells (Chen et al. 1992). Fetal tau comprises only the shortest isoform (0N3R); the other isoforms are added during brain development (Drubin and Kirschner 1986). In the human CNS, the six isoforms are present in roughly equal amounts; in particular, there is an equal balance of 3-repeat and 4-repeat isoforms (Goedert and Jakes 1990) that is perturbed in FTDP-17.
A notable feature of tau is its large number of potential phosphorylation sites, due to the frequency of S or T residues. Many of them (up to 17, depending on isoform) are part of SP or TP motifs and represent targets of proline-directed Ser/Thr protein kinases (e.g., MAP kinase, GSK-3^, cdk5, cdc2 etc. ). Other sites are targetted by a variety of kinases, including PKA, PKC, CaMK, SGCK, AKT, MARK, SAD and others (for reviews, see Chen et al. 2004; Stoothoff and Johnson 2005). Notably, the KIGS or KCGS motifs in the repeat domain (e.g, S262) are phosphorylated by MARK, which strongly reduces the tau-microtubule interactions (Biernat et al. 1993; note that the same phosphorylation also inhibits PHF aggregation, illustrating the analogous role of the repeat domain in physiological and pathological functions of tau; Schneider et al. 1999). A further potent detaching site is S214, which can be phosphorylated by PKA and is upregulated during mitosis (Brandt etal. 1994;Illenberger etal. 1998). Tau contains five Y residues (residues 18,29,197, 310,394), one of which (Y18) is phosphorylated by the Tyr-kinase Fyn (Lee et al. 2004a). Furthermore, tau contains one or two cysteines in the repeat domain (C291 in R2, C322 in R3) that can be engaged in intra- or intermolecular crosslinking, which affects conformation, dimerization, and aggregation (Schweers et al. 1995).
The main physiological function of tau, i.e., binding to microtubules, is achieved by the repeat domain and the adjacent proline-rich flanking domains. In general, 4-repeat tau binds microtubules more tightly (Butner and Kirschner 1991) whereas phosphorylation, especially in the repeat domain, tends to decrease the affinity (Biernat et al. 1993). Like soluble tau, microtubule-bound tau is mostly in a natively unfolded state (Fig. 2) andis, therefore, poorly visible by X-ray fiber diffraction or (cryo-) electron microscopy (Santarella et al. 2004; Al-Bassam et al. 2002). This is in strong contrast to other microtubule-interacting proteins, such as motor proteins, which show a periodic
Fig. 2. Model of microtubule protofilament with bound kinesin and Tau. The protofilament consists of alternating subunits of a- and p-tubulin (~ 450 residues each) arranged in a polar fashion ("plus" end pointing to the cell periphery). The head domain of kinesin, a microtubule-dependent motor protein (~ 350 residues), has the compact folding typical of most cytoplasmic proteins. By contrast, Tau is natively unfolded; its structure is unknown in detail and modelled here as a random chain. Note that tau occupies a much larger volume than kinesin or tubulin
Fig. 2. Model of microtubule protofilament with bound kinesin and Tau. The protofilament consists of alternating subunits of a- and p-tubulin (~ 450 residues each) arranged in a polar fashion ("plus" end pointing to the cell periphery). The head domain of kinesin, a microtubule-dependent motor protein (~ 350 residues), has the compact folding typical of most cytoplasmic proteins. By contrast, Tau is natively unfolded; its structure is unknown in detail and modelled here as a random chain. Note that tau occupies a much larger volume than kinesin or tubulin binding pattern to microtubules commensurate with the tubulin lattice (8-nm axial repeat; Santarella et al. 2004). However, it appears that tau binds in an extended fashion to the outer tips of microtubule protofilaments. When microtubules are disassembled by low temperature, tau stabilizes the ring-like disassembly products consisting of tubulin oligomers. Nevertheless, the binding of tau to microtubules must be highly dynamic since nuclear magnetic resonance (NMR) studies reveal a high mobility of most tau residues even in the bound state (Woody et al. 1983). Since tau and motor proteins both bind to the outer surface of microtubules, they compete for binding sites, explaining why tau can interfere with transport along microtubules, which leads to an inhibition of anterograde transport in axons (Stamer et al. 2002).
The conformation of tau in solution is unknown and presumably highly variable, as expected for a natively unfolded protein. An NMR analysis of secondary chemical shifts of the repeat domain reveals little secondary structure, except for some motifs of nascent ^-structure near the beginnings of R2, R3, R4 (Mukrasch et al. 2005). These coincide with the regions involved in PHF assembly (see below). Nevertheless, FRET studies reveal a global (average) paperclip-like folding of tau in solution that results in a close juxtaposition of the repeat domain with the C-terminal and N-terminal ends of the molecule (Jeganathan et al. 2006; Fig. 3). This conformation is reminiscent of the discontinuous epitopes of certain antibodies (Alz50, MC1) that recognize early stages of AD and are generated by folding the N-terminus of tau over the repeat domain (Carmel et al. 1996; Jicha et al. 1997).
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