**3.2 Structural stability of the tubulin-TauR2 complexes**

The all atom MD simulations were performed on tubulin-TauR2 complexes namely 6CVN-TauR2, 6CVN\*-TauR2, βI/α/βI-TauR2, βIIb/α/βIIb-TauR2, βIII/α/βIII-TauR2 using Gromacs 2018.1 [78]. The stability of these tubulin-TauR2 complexes is accessed by plotting the potential energy during the simulation period, which highlight that all the complexes are well minimized, and simulation trajectories are well converged during the simulation period of 100 ns (**Figure 3**).

The parameters describing the stability of tau-tubulin complex such as RMSD (root mean square deviation), RMSF (root mean square fluctuation), and R*g* (radius of gyration) was studied. The RMSD values for simulated tubulin-TauR2 complexes, tauR2 and backbone atoms of tubulin trimer without considering


#### **Figure 2.**

*Multiple sequence analysis of different* β*-tubulin isotypes. The* β*I,* β*IIb,* β*III tubulin isotypes and template 6CVN show maximum residue variations mainly at C-terminal tail region. The TauR2 binding regions H12 helix and C-terminal tail region of* β*-tubulin subunits are shown in hot pink and brown, respectively.*


#### **Table 2.**

*Validation of three-dimensional models generated for* β*I,* β*IIb and* β*III isotypes chain A and chain C using Swiss model GMQE score, Verify-3D, Errat score.*

disordered C-tail were plotted over the trajectory. This analysis reveals the stability of all the studied complexes throughout the simulation time i. e. 100 ns. **Figure 4A** and **B** shows the RMSD plot for studied tubulin-TauR2 complexes and TauR2, respectively. The RMSD for the complex βIII/α/βIII-TauR2 is observed to be relatively more stable than other tubulin-TauR2 complexes. Similarly, structure

**77**

has specificity towards tubulin subunits. s.

*Homology Modeling of Tubulin Isotypes to Investigate MT-Tau Interactions*

**Region** β**1 tubulin** β**2 tubulin** β**3 tubulin**

% of outlier 0.2 (1) 0.2 (1) 0.4 (2) 0 (0) 0 (0) 0.4 (2)

*Ramachandran plot showing the percentage of residues in the different regions for tubulin isotypes obtained* 

98.9 (444)

**Chain A Chain C Chain A Chain C Chain A Chain C**

1.3 (6) 0.9 (4) 0.7 (3) 1.1 (5) 1.1 (5) 0.7 (3)

98.9 (438)

98.9 (443)

98.9 (443)

98.9 (443)

of TauR2 bound to βIII/α/βIII tubulin trimer expresses stable dynamics during the simulation. The complex 6CVN-TauR2 is stabilized at higher RMSD values, the primary reason for this elevated RMSD value is absence of C-tail region which highlights the importance of C-terminal tail in the stabilizing tubulin-TauR2 complex. Average backbone RMSD value is converged at ~3.5 Å hence represents the equilibration of all above simulated systems (**Figure 5**). The molecular dynamics simulation movies reveals the stable dynamics of all the simulated systems 6CVN-TauR2, 6CVN\*-TauR2, βI/α/βI-TauR2, βIIb/α/βIIb-TauR2 and βIII/α/βIII-TauR2 (https://youtu.be/mU2Jrm5jusY, https://youtu.be/Sr2JiQWha9A, https://youtu.be/ U5S6X-o8kO8, https://youtu.be/xYbm9eCsE4Q, https://youtu.be/0H0CsmveT24) respectively. Further, specificity of TauR2 towards tubulin subunits was accessed by replacing the TauR2 with negative control 'polyA' peptide of same length. Interestingly, This system having negative control poly A bound to 6CVN\* shows the weak binding during the simulation. These weaker interactions of polyA peptide with tubulin subunits (https://youtu.be/ZEFQblQTHqk) represents that tauR2

*Energy Minimization Plot Potential energy over the simulation time plotted for 6CVN-TauR2 (black), 6CVN\*- TauR2 (orange),* β*I/*α*/*β*I-TauR2 (green),* β*IIb/*α*/*β*IIb-TauR2 (cyan),* β*III/*α*/*β*III-TauR2 (violet) are shown.*

*DOI: http://dx.doi.org/10.5772/intechopen.95792*

*from the Ramachandran plot using PROCHECK.*

98.4 (442)

% of most favored

% of additional allowed regions

regions

**Table 3.**

**Figure 3.**

*Homology Modeling of Tubulin Isotypes to Investigate MT-Tau Interactions DOI: http://dx.doi.org/10.5772/intechopen.95792*


#### **Table 3.**

*Homology Molecular Modeling - Perspectives and Applications*

disordered C-tail were plotted over the trajectory. This analysis reveals the stability of all the studied complexes throughout the simulation time i. e. 100 ns. **Figure 4A** and **B** shows the RMSD plot for studied tubulin-TauR2 complexes and TauR2, respectively. The RMSD for the complex βIII/α/βIII-TauR2 is observed to be relatively more stable than other tubulin-TauR2 complexes. Similarly, structure

*Validation of three-dimensional models generated for* β*I,* β*IIb and* β*III isotypes chain A and chain C using* 

*Multiple sequence analysis of different* β*-tubulin isotypes. The* β*I,* β*IIb,* β*III tubulin isotypes and template 6CVN show maximum residue variations mainly at C-terminal tail region. The TauR2 binding regions H12 helix and C-terminal tail region of* β*-tubulin subunits are shown in hot pink and brown, respectively.*

**S. No. Chains GMQE Verify3D Errat z-score** β1 (A) -subunit 0.98 93.13% 81.3212 −8.93 β1 (C) -subunit 0.98 92.02% 83.2569 −8.78 β2b (A) -subunit 0.98 98.43% 87.471 −8.65 β2b (C) -subunit 0.98 98.43% 83.2947 −8.37 β3 (A) -subunit 0.98 98.44% 86.3636 −8.54 β3 (C) -subunit 0.98 92.67% 83.33 −8.39

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**Table 2.**

*Swiss model GMQE score, Verify-3D, Errat score.*

**Figure 2.**

*Ramachandran plot showing the percentage of residues in the different regions for tubulin isotypes obtained from the Ramachandran plot using PROCHECK.*

**Figure 3.**

*Energy Minimization Plot Potential energy over the simulation time plotted for 6CVN-TauR2 (black), 6CVN\*- TauR2 (orange),* β*I/*α*/*β*I-TauR2 (green),* β*IIb/*α*/*β*IIb-TauR2 (cyan),* β*III/*α*/*β*III-TauR2 (violet) are shown.*

of TauR2 bound to βIII/α/βIII tubulin trimer expresses stable dynamics during the simulation. The complex 6CVN-TauR2 is stabilized at higher RMSD values, the primary reason for this elevated RMSD value is absence of C-tail region which highlights the importance of C-terminal tail in the stabilizing tubulin-TauR2 complex. Average backbone RMSD value is converged at ~3.5 Å hence represents the equilibration of all above simulated systems (**Figure 5**). The molecular dynamics simulation movies reveals the stable dynamics of all the simulated systems 6CVN-TauR2, 6CVN\*-TauR2, βI/α/βI-TauR2, βIIb/α/βIIb-TauR2 and βIII/α/βIII-TauR2 (https://youtu.be/mU2Jrm5jusY, https://youtu.be/Sr2JiQWha9A, https://youtu.be/ U5S6X-o8kO8, https://youtu.be/xYbm9eCsE4Q, https://youtu.be/0H0CsmveT24) respectively. Further, specificity of TauR2 towards tubulin subunits was accessed by replacing the TauR2 with negative control 'polyA' peptide of same length. Interestingly, This system having negative control poly A bound to 6CVN\* shows the weak binding during the simulation. These weaker interactions of polyA peptide with tubulin subunits (https://youtu.be/ZEFQblQTHqk) represents that tauR2 has specificity towards tubulin subunits. s.

#### **Figure 4.**

*Stability of the tubulin-TauR2 complex and TauR2. (A) The Root mean square deviation values (RMSD) for tubulin-tauR2 complexes. RMSD values for 6CVN, 6CVN\*,* β*I/*α*/*β*I,* β*IIb/*α*/*β*IIb and* β*III/*α*/*β*III have been plotted in black, orange, green, cyan and violet, respectively. (B) The Root mean square deviation values for TauR2 shown using same color Scheme as in (A).*

**Figure 5.** *Backbone Root mean square deviation for different tubulin subunits. Color scheme is same as Figure 3.*

#### **3.3 Residue fluctuations of tubulin subunits and TauR2**

The flexibility of tubulin trimers systems and TauR2 has been studied using RMSF analysis. For this analysis Cα-atom from the backbone was selected to get fluctuations in the overall protein. **Figure 6** represents the RMSF for tubulin

**79**

**Figure 6.**

*Figure 3.*

*Homology Modeling of Tubulin Isotypes to Investigate MT-Tau Interactions*

subunits and TauR2. The residues from the H12 helix of β-tubulin and the C-terminal tail region (residue 400–451) show significant decrease in the RMSF values, as their free dynamics is arrested by tau binding (**Figure 6A** and **B**). RMSF

*Root mean square fluctuations (RMSF) of different* β*/*α*/*β *tubulin subunits and TauR2 (A) RMSF of different*  β*/*α*/*β *tubulin subunits (B) Magnified view of their C-terminal H12 helix and tail regions (C) RMSF of TauR2 bound with different* β*/*α*/*β *tubulin subunits observed during the simulations5. Color scheme is same as* 

*DOI: http://dx.doi.org/10.5772/intechopen.95792*

*Homology Molecular Modeling - Perspectives and Applications*

**78**

**Figure 5.**

**Figure 4.**

*TauR2 shown using same color Scheme as in (A).*

**3.3 Residue fluctuations of tubulin subunits and TauR2**

The flexibility of tubulin trimers systems and TauR2 has been studied using RMSF analysis. For this analysis Cα-atom from the backbone was selected to get fluctuations in the overall protein. **Figure 6** represents the RMSF for tubulin

*Backbone Root mean square deviation for different tubulin subunits. Color scheme is same as Figure 3.*

*Stability of the tubulin-TauR2 complex and TauR2. (A) The Root mean square deviation values (RMSD) for tubulin-tauR2 complexes. RMSD values for 6CVN, 6CVN\*,* β*I/*α*/*β*I,* β*IIb/*α*/*β*IIb and* β*III/*α*/*β*III have been plotted in black, orange, green, cyan and violet, respectively. (B) The Root mean square deviation values for* 

subunits and TauR2. The residues from the H12 helix of β-tubulin and the C-terminal tail region (residue 400–451) show significant decrease in the RMSF values, as their free dynamics is arrested by tau binding (**Figure 6A** and **B**). RMSF

#### **Figure 6.**

*Root mean square fluctuations (RMSF) of different* β*/*α*/*β *tubulin subunits and TauR2 (A) RMSF of different*  β*/*α*/*β *tubulin subunits (B) Magnified view of their C-terminal H12 helix and tail regions (C) RMSF of TauR2 bound with different* β*/*α*/*β *tubulin subunits observed during the simulations5. Color scheme is same as Figure 3.*

values for the tubulin β-subunits in the systems 6CVN\*, βIIb/α/βIIb and βIII/α/ βIII are lesser than those of 6CVN and βI/α/βI tubulin subunits (**Figure 6B**). This observation also highlights the binding of TauR2 at the interdimer interface where residual fluctuations are less. However, the part of C-tail region which has no direct contact with TauR2 is highly flexible (**Figure 6B**). The H12 helix and C-terminal tail region of the tubulin subunits significantly contribute to the noncovalent interactions resulting towards stronger binding of TauR2. Therefore, these intermolecular interactions were analyzed in detail and are discussed in the section '*Intermolecular interactions between tubulin and tau'*. Further, atomic Cα-fluctuations of TauR2 (**Figure 6C**) was also studied for better understanding its conformational behavior during the MD simulations. It is surprising to observe highest fluctuations at the N- and C-terminal region in TauR2 bound to 6CVN, where the C-terminal tail region is absent (**Figure 6C**). Interestingly, residual fluctuations expressed by TauR2 bound to βIII/α/βIII-tau complex are much lesser as compared to 6CVN\*-TauR2, βI/α/βI-TauR2 and βIIb/α/βIIb-TauR2 complexes (**Figure 6C**). This also proves that the C-terminal tail region of tubulin subunits plays an important role in the binding of TauR2.

Overall, RMSF analysis suggests the significance of H12-helix and C-terminal tail region in stabilization of the microtubule by binding of tau repeats (TauR2) and it also reveals the greater affinity of TauR2 towards βIII tubulin isotypes which are overexpressed in neuronal cells and brain. Further compactness of all the tubulin-TauR2 complexes was explored by calculating the radius of gyration (*R*g) and this analysis is discussed in the next section.
