Figure 1.
Primary Sequence of 441-Residue Tau
(A) Domain organization of htau40. The isoform htau40 is the largest one in the human central nervous system (441 residues). Depending on isoform, the C-terminal half contains three or four pseudorepeats (∼31 residues each, R1–R4, cyan), which are involved in MT binding and form the core of PHFs. The two hexapeptides that are essential for aggregation of tau into PHFs are highlighted. P1 and P2 are the proline rich regions. Domain boundaries are labelled by the residue numbers.
(B) Amino acid sequence of htau40. Amino acids are colour coded according to their properties: basic (blue), non-polar (green), polar uncharged (brown), acidic (red). On top of the amino acid sequence, the domain organization is indicated. The two hexapeptides in repeats R2 and R3 are shown as black bars.
Figure 2.
441-Residue Tau at Single-Residue Resolution
(A) Number of intrinsically disordered proteins with NMR backbone assignment.
(B) 1H-15N HSQC spectra of htau40 with 1H projection on top.
(C) Comparison of spectral overlap observed in HSQC spectra of htau40 (solid line) and 731-residue malate synthase G (dashed line). Black, blue, and green indicate 15N chemical shift tolerances of 0.2, 0.15, and 0.1 ppm, respectively.
(D) Superposition of a selected portion of the 1H-15N HSQCs of the three tau fragments K25 (yellow), K32 (red), and K10 (green), and of 441-residue htau40 (blue). The domain organization of the three tau constructs is indicated.
(E) Assignment strategy for htau40. 2-D strips of high-resolution 3-D HCANNH (left) and HNN spectra (right). The connectivity path linking residues V306 to V309 is marked in green.
Figure 3.
Residual Secondary Structure of Tau in Solution
(A) Secondary chemical shifts for Cα. Regions of β-structure (α-helical) propensity are identified by negative (positive) values extending over several residues and are highlighted in yellow (red). Regions that preferentially populate polyproline II helix have negative Cα secondary chemical shifts and are marked in green. The domain organization of htau40 is shown above. Repeat boundaries are indicated by vertical dashed lines.
(B) Differences between experimental 3J(HNHα) scalar couplings and random coil values as a function of sequence number. Regions of β-structure (α-helical) propensity are identified by positive (negative) values extending over several residues. Regions that preferentially populate polyproline II helix have negative Δ3J(HNHα) values and can therefore readily be distinguished from β-structure.
(C) Cα secondary chemical shifts of regions with a propensity to adopt β-structure.
(D,E) Comparison of Cα secondary chemical shifts with Δ3J(HNHα) values in regions of transient helical structure. On top, the estimated population of α-helical structure is indicated. Mapping onto a helical wheel reveals two amphiphatic helices.
(F) Comparison of Cα secondary chemical shifts with Δ3J(HNHα) values in regions that transiently populate polyproline II helical conformations.
(G) Schematic representation of the elements of transient secondary structure in htau40: β-structure (yellow), α-helical (red), polyproline II (green). In case of β-structure propensity, only regions with populations of 17% or more are shown.
Figure 4.
RDCs Observed in Weakly Aligned Tau
(A) Profile of 1D(HN) dipolar couplings observed in htau40 aligned in Pf1 bacteriophage at 5 °C. Increased positive (for the β-structures and polyproline II helix regions) as well as negative (for the α-helices) RDCs indicate rigidity on the nano- to microsecond time scale.
(B,C) Comparison of 1D(HN) dipolar couplings (black bars) with Cα secondary chemical shifts (red line) in the turn region 345DKFD348 (B) and the helical region 428ladevsasla437 (C). Sign-inversion of RDCs indicates that the H-N internuclear vectors are parallel to the long axis of the corresponding segment.
Figure 5.
Robustness of Secondary Structure Propensity of Tau
(A) Superposition of Cα secondary chemicals shifts observed in 441-residue Tau (black) and in a 198-reside fragment (K32) comprising only the repeat domain and its flanking regions (shown as red bars).
(B) Correlation of Cα secondary chemicals shifts observed in htau40 at 278K and 293K.
Figure 6.
Plot of 15N R1ρ spin relaxation rates along the amino acid sequence. High R1ρ rates reflect increased rigidity on a pico- to nanosecond time scale and are mostly found in regions that transiently populate extended structures (β-structure or polyproline II helix).
Figure 7.
PRE of Amide Protons in Spin-Labelled Tau
(A–F) PRE profiles of amide protons in spin-labelled htau40. wt htau40 and five single-cysteine mutants (A15C, A72C, A239C, A384C, and A416C) were labelled with MTSL at residue (A) 15, (B) 72, (C) 239, (D) 291 and 322, (E) 384, and (F) 416. Intensity ratios were averaged over a three-residue window. Decreases in peak intensity ratios that occur far from the site of spin-labelling (>10 residues) are indicative of long-range contacts (<25 Å) between the spin-label and distant areas of sequence.
Figure 8.
Native-State Conformations of 441-Residue Tau in Solution
(A) Representation of the conformations of htau40 calculated from PRE data. Left panel: Colour coding follows the domain organization diagram shown above. Regions of transient α-helical structure (H1[114–123] and H2[428–437]) and β-structure (B2[274–284], B3[305–315] and B4[336–345]) are shown in red and yellow, respectively. Polyproline II stretches (PPP[175–184], PP[216–223], and P[232–239]) are coloured green. In the background, an ensemble of 20 conformations is shown. Right panel: Same conformation as in left panel, but colour coding according to the domain organization of tau.
(B) Average contact map for the seven lowest-energy structures obtained from a calculation in which all distance restraints were enforced onto a single molecule. A continuous grey scale from 3 Å (black) to 22 Å (white) is used.
Figure 9.
Influence of Ionic Strength and Urea on Intramolecular Long-Range Interactions
(A) Hydrodynamic radius values of htau40 in buffer (99.9 % D2O, 50 mM phosphate buffer [pH 6.9]), upon addition of 600 mM NaCl and in the presence of 8 M urea.
(B) PRE profiles of amide protons in spin-labelled htau40 in buffer (red bars) and in the presence of 600 mM NaCl (blue bars). The single-cysteine mutant A239C was labelled with MTSL. Intensity ratios were averaged over a three-residue window.
Figure 10.
(A) Superposition of a selected portion of the 1H-15N HSQC spectra of htau40 in the free state (blue) and in the presence of MTs (red). Resolved peaks are labelled with their assignments. The tau:tubulin heterodimer ratio was 2:1. To highlight changes in NMR signal intensities, a trace through the center (solid line) of selected peaks is shown.
(B) NMR signal intensity ratios between signals observed for htau40 in the MT-bound and in the free state. Repeat boundaries are indicated by vertical dashed lines. Hot spots of binding are highlighted in blue.
(C) Absolute magnitude of 15N chemical shift changes. The estimated error based on 1H-15N HSQCs of two different htau40 samples was 0.025 ppm.
(D,E) NMR signals intensity ratios in the proline-rich regions (D) and in the repeat domain (E). Values were taken from (B). In (E), the position of the two hexapeptides is indicated by black bars.
Figure 11.
Comparison of MT-Binding Profile with Hydrophobicity Pattern of Tau
(A) NMR signal intensity ratios between signals observed for htau40 in the MT-bound and in the free state (see also Figure 10B) are shown as stars and connected by lines. Hydrophobicity values calculated according to the Kyte-Doolittle scale are shown as green bars. Hydrophobic regions have positive or small negative hydrophobicity values. The location of transient secondary structure observed in unbound htau40 (i.e., as monomer in solution) is indicated as schematic on top.
In (B) and (C) the regions that are important for interaction with MTs are shown in detail. The amino acid sequence is shown below. It is colour coded according to Figure 1B. The regions of tau that transiently populate β-structure and polyproline II helix in the unbound state are marked by yellow arrows and green bars. Phosphorylation sites are marked by yellow circles.