4 DISCUSSION
4.5.1 NMR approaches identified the potential binding site
Our Tau-MT/tubulin interaction studies showed clear evidence that the different binding domains bind independent of each other. To confirm whether all these binding hot spots or active sites bind at a single binding pocket or not and also to see where does it bind different competition experiments were performed. The attempt to obtain further insight into the tubulin binding site of the Tau binding domains a combination of methods were used.
4.5.1.1 Tau-MT interaction involves interaction with C-terminal domain of tubulin
The titration of hTau40 with the C-terminal peptides of tubulin (section 3.20.1) suggests that the positively charged MT binding region of Tau can transiently interact with the negatively charged C-terminus of tubulin. The similarities in the chemical shift profiles with that of hTau40 in presence of MTs (Figure 16, Figure 56) underline the affected regions with this interaction. By using recombinant alpha and beta-tubulin C-terminal fragments, the involvement of both of these tubulin regions in Tau binding had been suggested previously (Devred, Barbier et al. 2004, Lefevre, Chernov et al. 2011).
The data suggest that neutralization of the electrostatic field generated by the negatively charged C-terminus of tubulin is required for MT-assembly, in line with the observation
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that removal of the C-terminus of tubulin by subtilisin promotes MT-assembly (Serrano, Avila et al. 1984).
4.5.1.2 MT targeting drug Vinblastine and hTau40 compete for binding
We further used different tubulin drugs with known binding sites (Figure 57), which are classified into different categories based on their activities and functions (section 1.3.6). These compounds were allowed to compete with hTau40 or Tau peptides for binding with MT/Tubulin. Initially we performed the competition experiments of full length Tau with MT targeting drugs such as Taxol, Baccatin, Thalidomide, Colchicine and Vinblastine (section 3.20.2).
Titration of each MT drug was carried out in increasing amounts of concentrations to a solution of 15N-labeled hTau40 bound to MTs and the variation in the NMR signal line broadening caused by MT interaction in specific regions of Tau was observed. None of the compounds other than Vinblastine was found to be competing with Tau for MT binding (Figure 58 and Figure 59). The decrease in NMR line broadening is as a result of the impairing of Tau-MT complex as Vinblastine depletes Tau off from tubulin/MT binding pocket and more Tau becomes free in solution. Hence line broadening is less compared to the isolated Tau-MT complex. The competition experiments were performed with unpolymerized tubulin in the similar manner using Vinblastine, Taxol and Thalidomide, where only Vinblastine showed tendency to compete with Tau (Figure 60).
Taxol, Baccatin, Colchicine and Thalidomide were found to be inert to compete with Tau for tubulin/MT binding. The fact that Vinblastine competes with Tau bound to MT as well as tubulin shows the mutual dependence of Tau and Vinblastine for binding at a single binding site on MT.
4.5.1.3 STD NMR method revealed the potential binding site of Tau on MTs
To further verify the competition observed between Tau and vinblastine 1D STD NMR based competition experiments were performed with all the peptides used in the structural investigation. The STD spectra of each peptide bound to tubulin indicated the specific residues involved in binding, which are part of the structured region of these peptides in the MT-bound state (section 3.6). Moreover the residues that constituted the structured region of the peptide showed highest STD effects showing their contact with
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tubulin. All the peptides were allowed to compete with the compounds and the STD NMR based competition experiments performed are listed in the Table 9. The complete analysis of the STD spectra of all the combinations of peptides and MT drugs shows that only vinblastine is competing with the Tau binding peptides (Figure 61) and the relative effect is different for different peptides (Figure 62) and this suggests that TR3 and TP2 are having highest affinity for MT/tubulin binding. The similar observation was found when vinblastine was competing with MT-bound Tau peptides. The variation in intensities of different resonances in the spectrum is different depending on the residues which are nearby tubulin. This is possible only if the ligands are coming in the close proximity to share the same binding site. The extent of competition is found to be prominent between TR3 peptide and vinblastine where the STD signal intensities of TR3 is reduced by 60%, where the Y310 aromatic side chain proton intensities were compared in the presence and absence of MT drugs. This strongly supports the competition between hTau40 and Vinblastine for MT/tubulin binding (Figure 61 and Figure 63). Till now it is evident that Tau and vinblastine target the same binding site or their binding is mutually dependent.
4.5.1.4 I19L peptide from the N-terminal stathmin like domain compete with Tau peptides
The conflict in these findings is the competition between MT stabilizing and destabilizing agents. Tau as a MT stabilizing agent is found to be competing with vinblastine for binding either to tubulin or MTs. The characteristic feature of vinblastine as a MT destabilizing agent is it induces the formation of spiral tubulin aggregates (Weisenberg and Timasheff 1970, Himes 1991) whereas Tau promotes the formation of straight protofilaments. To further support the possibility for Tau to share the vinblastine binding site, the I19L peptide belongs to the N-terminal part of the stathmin like domain (Clément, Jourdain et al. 2005) was allowed to compete with Tau peptides. The reason behind this is that I19L was shown to be binding on α-tubulin at the longitudinal tubulin interdimer interface between the helix 10 (H10) and β-strand S9 of the crystal structure determined for tubulin-colchicine/RB3-SLD crystal (Clément, Jourdain et al.
2005). This is very close to the vinblastine binding site which also binds on α-tubulin at the tubulin interdimer interface. The STD based competition experiments revealed that Tau peptides compete with I19L as observed in the case of Vinblastine (Figure 65a and
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b). Finally Vinblastine showed competitive binding with I19L (Figure 65c). It is to be noticed that the repeat domains of hTau40 have sequence identities and similarities (Figure 64) and hence the competition observed between I19L and Tau fragments is in line with the sequence similarity.
It is highly important that Vinblastine being a MT destabilizing agent compete with MT stabilizing protein Tau. It was shown that the negative regulator of MT dynamics shows competition with the stabilizing agents. A similar observation was found while characterizing the binding site of antimitotic drug Tubulysin, a microtubule destabilizing agent, it shared the binding site of Epothilone A, which is a microtubule stabilizing agent (Kubicek, Grimm et al.).