Mobility and Core-Protein Binding Patterns of Disordered C-Terminal Tails in β-Tubulin Isotypes

Abstract

Although they play a significant part in the regulation of microtubule structure, dynamics, and function, the disordered C-terminal tails of tubulin remain invisible to experimental structural methods and do not appear in the crystallographic structures that are currently available in the Protein Data Bank. Interestingly, these tails concentrate most of the sequence variability between tubulin isotypes and are the sites of the principal post-translational modifications undergone by this protein. Using homology modeling, we developed two complete models for the human αI/βI- and αI/βIII-tubulin isotypes that include their C-terminal tails. We then investigated the conformational variability of the two β-tails using long time-scale classical molecular dynamics simulations that revealed similar features, notably the unexpected presence of common anchoring regions on the surface of the tuulin dimer, but also distinctive mobility or interaction patterns, some of which could be related to the tail lengths and charge distributions. We also observed in our simulations that the C-terminal tail from the βI isotype, but not the βIII isotype, formed contacts in the putative binding site of a recently discovered peptide that disrupts microtubule formation in glioma cells. Hindering the binding site in the βI isotype would be consistent with this peptide's preferential disruption of microtubule formation in glioma, whose cells overexpress βIII, compared to normal glial cells. While these observations need to be confirmed with more intensive sampling, our study opens new perspectives for the development of isotype-specific chemotherapy drugs.

An Emerging Role for Tubulin Isotypes in Modulating Cancer Biology and Chemotherapy Resistance

“…The intermediate domain of the tubulin body has been proposed to regulate microtubule dynamics through its conformational state and by coupling the tubulin conformation to the tubulin GTP hydrolysis cycle [ 99 , 100 ]. In addition, the isotype defining region of the tubulin proteins, the C-terminal tail, which regulates microtubule dynamics through interactions with the intermediate domains of neighbouring tubulin isotypes to destabilise microtubules [ 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 ] may also contribute to the dynamic features of microtubules through differences in the conformational space covered by these flexible regions of the protein and by interacting with neighbouring tubulin heterodimers in an isotype-specific manner [ 101 , 102 , 103 , 116 ]. While the disordered nature of the tubulin C-terminal tail has hindered direct experimental characterisation of tail-body interactions proposed by these molecular dynamics simulations, evidence that the C-terminal tail region differentially affects microtubule assembly between isotypes [ 117 ] supports the notion that this region of the protein may significantly contribute to isotype-specific microtubule dynamics.…”

The neurofilament derived-peptide NFL-TBS.40-63 enters in-vitro in human neural stem cells and increases their differentiation

“…Indeed, the mitochondria network is closely linked to the proliferation mechanism thanks to the aspartate synthesis [ 39 ], and thus the peptide at low concentration could affect the mitochondria in hNSCs inducing a reduction in proliferation. Moreover, as previously mentioned, the peptide is derived from the tubulin binding site of neuron-specific cytoskeleton protein (neurofilament), which suggests that the peptide has more affinity with neuron-specific tubulin (like βIII tubulin) than other tubulins as previously shown [ 32 ]. Since all sequences of β-tubulin are very similar [ 40 ], it is possible than the NFL-TBS.40-63 peptide binds other tubulins with less affinity than with the βIII tubulin, resulting in modification of proliferation without affecting the microtubule network.…”

“…Moreover, similar docking experiments showed that the conformation of the β-tubulin C-terminal domain is important for the binding of the peptide. Indeed, the C-terminal region of βI tubulin, but not of βIII tubulin, forms internal contacts preventing the binding of the peptide on tubulin [ 32 ]. Finally it was shown that β-tubulins are differentially expressed depending on the tissue [ 33 ].…”

β-Tubulin carboxy-terminal tails exhibit isotype-specific effects on microtubule dynamics in human gene-edited cells

“…The paclitaxel binding site is located within the tubulin body, but structural deviations in the microtubule lattice indirectly induced by the C-terminal tail may affect the ability of paclitaxel to interact with these sites ( Nogales et al, 1995 ; Freedman et al, 2009 ). The unique electrostatic character of the βIII-tubulin C-terminal tail and, in particular, its terminal lysine residue may enable differential interaction of this C-terminal tail with the intermediate domain of neighboring α-tubulin subunits compared with the βI-tubulin C-terminal tail ( Sherman et al, 1983 ; Szasz et al, 1986 ; Mejillano et al, 1992 ; Pal et al, 2001 ; Freedman et al, 2011 ; Laurin et al, 2017 ). In addition, molecular dynamics simulations of βI- and βIII-tubulin C-terminal tail conformations suggest that these two tails differ in their contact with the β- and α-tubulin surface residues located principally in the intermediate domains of these proteins ( Downing & Nogales, 1999 ; Laurin et al, 2017 ).…”

“…The unique electrostatic character of the βIII-tubulin C-terminal tail and, in particular, its terminal lysine residue may enable differential interaction of this C-terminal tail with the intermediate domain of neighboring α-tubulin subunits compared with the βI-tubulin C-terminal tail ( Sherman et al, 1983 ; Szasz et al, 1986 ; Mejillano et al, 1992 ; Pal et al, 2001 ; Freedman et al, 2011 ; Laurin et al, 2017 ). In addition, molecular dynamics simulations of βI- and βIII-tubulin C-terminal tail conformations suggest that these two tails differ in their contact with the β- and α-tubulin surface residues located principally in the intermediate domains of these proteins ( Downing & Nogales, 1999 ; Laurin et al, 2017 ). Whereas the C-terminal tails are too disordered to resolve by cryo-EM, microtubules composed of βIII-tubulin have subtle progressive deviations in the microtubule fiber structure ( Vemu et al, 2016 ), and these may be influenced by interactions between the tail and tubulin body.…”