Tubulin proteins exist in the cell as dimers of α- and β-tubulin, two closely related proteins whose sequences essentially differ at the level of their disordered C-terminal tails; both tails bear a net negative charge but they differ in length and amino-acid composition. α,β-tubulin dimers are the building blocks of microtubules (MT), the largest components of the cytoskeleton, that form highways for intracellular trafficking as well as separating chromosomes during meiosis. Modeling and NMR studies have shown that in tubulin dimers, both α - and β-tails can interact with the structurally organized region of the protein dimer (the core region) in spite of the core surface potential being mainly negative [95
]. The tails are also known to contribute to the formation of microtubules by favoring the proper uptake of new tubulin dimers within the tubular architecture: alternative association forms of tubulin could be observed in the absence of tails [123
]. It is therefore likely that the MT tubulin tails interact with free tubulin dimers during the assembly process, thus orienting the dimers toward the desired binding geometry. This association however needs to be transient, since a large fraction of the tails (notably the longer β-tails) are released during the process and become free to interact with microtubule-binding proteins (MAPs) [121
]. Similar process has been observed by AFM when fibrin proteins assemble into fibrinogen [120
], while the C-terminal tails of RecA proteins have been shown to be involved in their association process into filaments [124
]. These observations indicate that the ability of the disordered protein tails to bind the protein core surface but also to unbind from it is key to their function.
How exactly the tails influence auto-assembly remains to be established. Theoretical simulation of the tubulin tails binding to their associated dimeric protein cores enabled to gain insights on this question [95
]. Notably, while the surface spanned by the tails during atomic molecular dynamics simulations was found compatible with ensemble observations obtained by AFM (radius of gyration), the simulation enabled proposing a finer characterization of the spatial and temporal distribution of the tails, based on specifically developed metrics using the position of the tail center of mass, together with time analysis of the contacts between tails and protein cores. This analysis revealed the presence of a handful of specific tail-binding spots, or anchors, distributed on the tubulin surface and presenting reduced surface areas. The tails develop versatile interactions with these binding spots, mostly based on electrostatic complementarity [95
]. Interestingly, negatively charged amino-acid patches distributed along the whole β-tail (see Figure 1
) can individually bind separate binding spots, and that adjacent negative patches can slide within a given anchor and exchange their binding interactions. Binding different sites on the core surface does not seem to be cooperative but rather self-exclusive, one reason being that several negative patches on the tail may not be able to simultaneously access spatially separated anchors. Another factor arises from the electrostatic potential around the tubulin dimer. Indeed, the electrostatic potential partitions the space available to the tails into electronegative regions, that are strongly repulsive for the most part of the tail length, and electropositive funnels that strongly attract the negative tail patches. This situation creates tension and frustration in the bound tails, part of which needs to reside in an unfavorable, repulsive region to allow contacts to form on the tubulin surface [95
]. Frustration, a tradeoff between conflicting forces within their interatomic contact network environment [125
], has been identified as a critical property of IDPs or IDRs binding to their protein targets [13
]. Because of unsolved conflicts at such interfaces, added to the multiplicity of binding sites, the disordered regions are prone to switching to alternate binding geometries. The concept of frustration extends to long-range interactions such as the response of protein tail conformations to the potential energy created by the protein core, coupled to the physical attachment between the tail and the protein core. Long range frustration may constitute a powerful driving force to facilitate the tail unbinding from its core protein. It is also easily tunable via changes in the salt concentration or modification of the charge distribution in the tail via post-translational modifications (PTM). Indeed, recent work from Bigman and Levy showed that PTMs tune the binding ability of the tails to the MT, a function that is also partly linked to their exclusion volume properties [121
]. Recent simulations of the hepatitis B virus (HBV) Core protein, that exhibits a 33-residue long, positively charged and intrinsically disordered C-terminal tail, suggests the existence of long range frustration in the binding of the negatively charged extremity of the tail to the positively charged extremity of the HBV capsid spike, with very sparse interactions between the rest of the tail and the external surface of the spikes [126
Figure 1. (Left) Negative surface electrostatic potential (-1 kT, magenta) of the αI/βIII isotype tubulin body without tails. The anchor residues involved in interactions with the disordered tail during molecular dynamics simulations are shown in blue; the representation is based on data published in (Laurin et al., Biochemistry 2017, 56, 1746); (right) schematic sequence of the αI/βIII isotype of tubulin, the acidic amino acids are highlighted in magenta and the basic terminal residue in green.