Thermodynamics of the Op18/Stathmin-Tubulin Interaction

Abstract

Op18/stathmin (stathmin) is an intrinsically disordered protein involved in the regulation of the microtubule filament system. One function of stathmin is to sequester tubulin dimers into assembly incompetent complexes, and recent studies revealed two tubulin binding sites per stathmin molecule. Using high sensitivity isothermal titration calorimetry, we document that at 10°C and under the conditions of 80 mM PIPES, pH 6.8, 1 mM EGTA, 1 mM MgCl 2 , 1 mM GTP these two binding sites are of equal affinity with an equilibrium binding constant of K 0 ‫؍‬ 6.0 ؋ 10 6 M ؊1 . The obtained large negative molar heat capacity change of ⌬C p 0 ‫؍‬ ؊860 cal mol ؊1 K ؊1 (referring to tubulin) for the tubulinstathmin binding equilibrium suggests that the hydrophobic effect is the major driving force of the binding reaction. Replacing GTP by GDP on ␤-tubulin had no significant effect on the thermodynamic parameters of the tubulin-stathmin binding equilibrium. The proposed pH-sensitive dual function of stathmin was further evaluated by circular dichroism spectroscopy and nuclear magnetic resonance. At low temperatures, stathmin was found to be extensively helical but devoid of any stable tertiary structure. However, in complex with two tubulin subunits stathmin adopts a stable conformation. Both the stability and conformation of the individual proteins and complexes were not significantly affected by small changes in pH. A 4-fold decrease in affinity of stathmin for tubulin was revealed at pH 7.5 compared with pH 6.8. This decrease could be attributed to a weaker binding of the C terminus of stathmin. These findings do not support the view that stathmin works as a pH-sensitive protein.

A designed ankyrin repeat protein selected to bind to tubulin caps the microtubule plus end

“…The D1-binding site does not overlap with that of RB3-SLD, is exclusively located on β-tubulin, and is distant by more than 40 Å from the α-β interface where the main differences between curved and straight tubulin are located. Taken together with the similar affinities of D1 for GDP-and GTP-tubulin and with a range of other biochemical evidence (11,33), our structural data further support the notion that GTP-tubulin in solution is curved similarly to GDP-tubulin and straightens only as it incorporates in microtubules. This result 2 (marked with an *) is an average of (+)-and (−) end growth velocity, because the two ends could not be distinguished due to similar growth velocities in this condition.…”

Stathmin/Op18 is a novel mediator of vinblastine activity

“…Fractions containing stathmin were then pooled and dry-lyophilized. Stathmin was resuspended and its concentration determined by the Lowry method with DC Protein Assay (Bio-Rad) and then adjusted, after ITC experiments, to reach the expected stathmin:tubulin stoichiometry of 0.5 [16].…”

“…Experimental temperature was chosen to maximize DH values and to compare our results with previously published data [16]. Tubulin concentrations in the calorimetric cell ranged from 5 to 20 lM, whereas the ligand (VLB or stathmin) concentrations varied from 50 to 200 lM.…”

“…A microcalorimetric approach allows the full characterization of this interaction in solution from a thermodynamic point of view [16]. We first investigated stathmin binding to tubulin in Fig.…”

Unbiased Proteomic and Transcript Analyses Reveal that Stathmin-1 Silencing Inhibits Colorectal Cancer Metastasis and Sensitizes to 5-Fluorouracil Treatment

“…In this respect, the N-terminal structure could determine affinity and accessibility of STMN1 interactions, because the majority of phosphorylation sites reside in the N-terminus. It was also suggested that N-terminal STMN1 may be intrinsically unstructured, and the molecule could depend on phosphorylation-induced conformation changes for activation (23).…”