Competition of Tau with MT drugs for binding

Interpretation of the titration with the C-terminal tubulin peptides is difficult as these are isolated peptides. More solid approach is the use of the MT drugs. The well characterized MT drugs are classified into four different classes on the basis of their stabilizing/destabilizing properties as well as their binding sites. The MT drugs used in our study include Taxol (Paclitaxel), Baccatin, (-)-Thalidomide, Colchicine and Vinblastine. The binding regions of these compounds are highlighted in Figure 57.

Competition experiments were done by adding the MT drugs into the MT or Tau-tubulin complex and variation in the signal intensities of 2-D ¹H-¹⁵N HSQC spectra was analysed.

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Figure 57: Binding sites of MT drugs. The binding sites of vinblastine (VB), colchicine (COL), taxol (PTX) and thalidomide (THAL) are indicated in the crystallographic structure of T₂R complex consisting of two tubulin heterodimers, vinblastine, colchicine and RB3 protein-stathmin like domain. The bound colchicine and vinblastine are shown using the stick model and a few residues involved in taxol binding are highlighted in pink color near the M-loop. (Figure modified from PDB 1Z2B) (Gigant, Wang et al.

2005).

3.8.2.1 Competition experiment with Vinblastine and hTau40

Vinblastine binds on α-tubulin, in between two tubulin heterodimers and inhibits MT assembly, instead it promotes the tubulin dimers to form curved protofilaments (Gigant, Wang et al. 2005). The possibility for vinblastine and Tau to share the same binding site was explored though competition experiments monitored by 2-D ¹H-¹⁵N HSQC spectra.

Vinblastine (VB) was allowed to interfere with Tau for MT binding by titrating it against the Tau-MT complex up to a concentration 20 times higher than Tau. It was observed that the line broadening in the HSQC spectrum of Tau-MT complex is reduced upon addition of VB. The successive addition of vinblastine separated Tau from MTs and led to an ~80% gain in the intensity (Figure 58a). Repeating these experiments with unpolymerized tubulin results in the similar observation (section 3.8.2.6). Hence the result supports the possibility for Tau and vinblastine to bind at the same or nearby binding site as the binding is influenced by each other.

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Figure 58: The competition of hatu40 and MT drugs vinblastine (a) and Thalidomide (b) are shown as the variation in the intensity ratios observed in the 2-D ¹H-¹⁵N HSQC spectra of MT-bound state. Black bars represent the line broadening of specific residues in hTau40 in the MT-bound state and grey lines shows the residual signal intensity of MT-bound hTau40 in the presence of compounds. The elevation in the intensity after the addition of vinblastine shows competition with hTau40 for MT binding as a result of impairing of hTau40 from MT.

3.8.2.2 Competition experiment with Thalidomide and hTau40

(-)-Thalidomide promotes MT assembly by binding to the side pocket on β-tubulin (Amos 2011), which is accessible from the outer surface of MTs. Addition of thalidomide in to the Tau-MT complex has no influence on the line broadening of hTau40 resonances in the ¹H-¹⁵N HSQC spectra (Figure 58b) and it was concluded that there is no competition between hTau40 and thalidomide for tubulin binding.

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3.8.2.3 Competition experiment with Baccatin and hTau40

Baccatin is another taxoid, an alternative for taxol and has higher solubility than taxol in water. Addition of baccatin in variable concentrations upto 20 times excess of hTau40 to the Tau-MT complex has no influence on the line broadening of hTau40 resonances in the 1H-15N HSQC spectra as shown in Figure 59a. The result again ruled out the possibility of Tau to bind near the M-loop and taken together competition experiments with taxol and baccatin rules out the possibility of Tau to bind at the luminal binding site

3.8.2.4 Competition experiment with Colchicine and hTau40

Colchicine is responsible for the inhibition of MT assembly binding within the heterodimer between α and β-tubulin in a pocket near the non-exchangeable GTP(Nogales, Wolf et al. 1998). Addition of colchicine in variable concentrations upto 20 times excess of hTau40 to the Tau-MT complex has no influence on the line broadening of hTau40 resonances in the 1H-15N HSQC spectra (Figure 59b). It indicates that Tau-MT interaction is not mediated by colchicine binding site.

3.8.2.5 Competition experiment with Taxol and hTau40 for MT binding

Taxol is one of the MT stabilizing drugs that bind in the luminal pocket (Nogales, Wolf et al. 1998). It has been believed that Tau and other MAPs stabilize MTs by binding at the taxol binding site near the M-loop of β-tubulin(Amos 2004). To verify the previous findings we performed the competition experiment using 2-D ¹H-¹⁵N HSQC experiments where taxol was allowed to interfere with Tau for MT binding. Titrating different fractions of taxol to the Tau-MT complex didn’t affect the Tau-MT binding as it was observed in the line broadening profile shown in Figure 59c. By increasing the concentration of Taxol, there is no competition with hTau40 as the signal intensities of hTau40 remain unchanged. The experiments using Tau-tubulin complex also provided the same result excluding the possibility of Tau to bind in the M-loop on β-tubulin.

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Figure 59: Inertness of Tau to compete with Baccatin, Colchicine and Taxol. No competition is observed as evident from the 2-D ¹H-¹⁵N HSQC spectra of MT-bound hTau40 with Baccatin (a), Colchicine (b) and Taxol (c) for MT-binding. The intensity ratio of MT-bound hTau40 is shown as black bars and grey line shows the residual signal intensity of MT-bound hTau40 in the presence of compounds.

It is evident from the competition experiments using hTau40 and MT drugs that only vinblastine shows clear competition for tubulin/MT binding.

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3.8.2.6 Vinblastine compete with hTau40 to bind to tubulin

Repeating the competition experiments between Vinblastine and hTau40 to bind to unpolymerized tubulin resulted in the similar observation found in the presence of MTs (section 3.8.2.1). Hence the result (Figure 60) supports the possibility for Tau and vinblastine to bind at the same or nearby binding site as the binding is influenced by each other.

Figure 60: Vinblastine competes with hTau40 for tubulin binding. Variation in the intensity ratios observed in the 2-D ¹H-¹⁵N HSQC spectra of hTau40 in the tubulin-bound state. Black bars represent the line broadening of specific residues in hTau40 in the tubulin-bound state and red, green and purple bars represent the residual signal intensity of tubulin-bound hTau40 in the presence of 5, 10 and 20 fold excess of vinblastine. The elevation in the intensity after the addition of vinblastine shows competition with hTau40 for tubulin binding.

All these data are in line with the presence of a single major binding site for Tau on MTs. At this point the question is where all these Tau domains bind. Initial competitions showed vinblastine compete and showed significant effect. To further investigate the presence of a common binding site for Tau and vinblastine we combined the results from competition experiments and final confirmation by NMR was done with the help of STD NMR experiments.

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