Probing the native structure of stathmin and its interaction domains with tubulin - Combined use of limited proteolysis, size exclusion chromatography, and mass spectrometry

Stathmin is a cytosoluble phosphoprotein proposed to be a regulatory relay integrating diverse intracellular signaling pathway. Its interaction with t ubulin modulates microtubule dynamics by destabilization of assembled micro tubules or inhibition of their polymerization from free tubulin. The aim of this study was to probe the native structure of stathmin and to delineate its minimal region able to interact with tubulin. Limited proteolysis of st athmin revealed four structured domains within the native protein, correspo nding to amino acid sequences 22-81 (I), 95-113 (II), 113-128 (III), and 12 8-149 (IV), which allows us to propose stathmin folding hypotheses. Further more, stathmin proteolytic fragments were mixed to interact with tubulin, a nd those that retained affinity for tubulin were isolated by size exclusion chromatography and identified by matrix-assisted laser desorption/ionizati on time-of-flight mass spectrometry. The results indicate that, to interact with tubulin, a stathmin fragment must span a minimal core region from res idues 42 to 126, which interestingly corresponds to the predicted alpha-hel ical "interaction region" of stathmin, In addition, an interacting stathmin fragment must include a short N- or C-terminal extension. The functional s ignificance of these interaction constrains is further validated by tubulin polymerization inhibition assays with fragments designed on the basis of t he tubulin binding results, The present results will help to optimize furth er stathmin structural studies and to develop molecular tools to target its interaction with tubulin.