Abstract
αβ-tubulin dimers need to convert between a ‘bent’ conformation observed for free dimers in solution and a ‘straight’ conformation required for incorporation into the microtubule lattice. Here, we investigate the free energy landscape of αβ-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Specifically, we performed molecular dynamics, potential-of-mean force simulations to obtain the free energy profile for unpolymerized GDP-bound tubulin as a function of the ∼12° intradimer rotation differentiating the straight and bent conformers. Our results predict that the unassembled GDP-tubulin heterodimer exists in a continuum of conformations ranging between straight and bent, but, in agreement with existing structural data, suggests that an intermediate bent state has a lower free energy (by ∼1 kcal/mol) and thus dominates in solution. In agreement with predictions of the lattice model of microtubule assembly, lateral binding of two αβ-tubulins strongly shifts the conformational equilibrium towards the straight state, which is then ∼1 kcal/mol lower in free energy than the bent state. Finally, calculations of colchicine binding to a single αβ-tubulin dimer strongly shifts the equilibrium toward the bent states, and disfavors the straight state to the extent that it is no longer thermodynamically populated.
Highlights
Microtubules (MTs) are dynamic cytoskeletal polymers formed by the polymerization of ab-tubulin, a globular heterodimer comprised of two structurally related 55 kDa a- and b-subunits
Tubulin has been shown to exist in two extreme conformations: a ‘‘straight’’ conformation observed in antiparallel zinc-induced tubulin sheets [2,3,4,5,6], which is compatible with incorporation into the MT lattice, and a ‘‘bent’’ conformation observed in the structure (T2R complex) of tubulin bound with colchicine, a MT-destabilizing drug, in a complex with the stathmin-like domain of RB3 (SLD-RB3) [7]. (The resolution of this T2Rcolchicine structure was recently enhanced to 2.73 A [8].) X-ray crystallographic studies have shown bent tubulin structures in a complex with SLD-RB3 [9], bound to other MTdestabilizing drugs, as well as MT-stabilizing drugs in a complex with tubulin tyrosine ligase (TTL) [10]
We find it geometrically infeasible for both tubulins to be in a fully bent state, both can exist in an intermediately bent state when laterally associated, and this state represents,25% of the population. (The two tubulins are constrained to maintain the same intradimer angle across the potential of mean force’’ (PMF), i.e., we have not investigated laterally associated straight-bent pairs, geometrically we expect these to be unfavorable.) Overall, comparison of the ‘free’ and laterally-paired tubulin PMFs suggests that lateral association shifts the bending equilibrium by,1.5 kcal/mol toward the straight conformation, providing direct support for the lattice model of microtubule assembly
Summary
Microtubules (MTs) are dynamic cytoskeletal polymers formed by the polymerization of ab-tubulin, a globular heterodimer comprised of two structurally related 55 kDa a- and b-subunits. We investigate the free energy landscape of ab-tubulin using molecular dynamics simulations, emphasizing implications for models of assembly, and modulation of the conformational landscape by colchicine, a tubulin-binding drug that inhibits microtubule polymerization. Tubulin has been shown to exist in two extreme conformations: a ‘‘straight’’ conformation observed in antiparallel zinc-induced tubulin sheets [2,3,4,5,6], which is compatible with incorporation into the MT lattice, and a ‘‘bent’’ conformation observed in the structure (T2R complex) of tubulin bound with colchicine, a MT-destabilizing drug, in a complex with the stathmin-like domain of RB3 (SLD-RB3) [7]. In addition a ,12u intradimer rotation is required to superimpose both subunits of the straight and bent tubulin structures.
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