Microtubule +TIP Protein EB1 Binds to GTP and Undergoes Dissociation from Dimer to Monomer on Binding GTP

Abstract

The +TIP protein EB1 autonomously tracks the growing plus end of microtubules and regulates plus-end dynamics. Previous studies have indicated that EB1 can recognize GTP-bound tubulin structures at the plus end, and it localizes on the microtubule surface at a site close to the exchangeable GTP-binding site of tubulin. Although the GTP-dependent structural change in tubulin has been demonstrated to be a critical determinant for recognition of plus ends by EB1, the effect of GTP on the structure of EB1 has remained unclear. Here, we have used spectroscopic, calorimetric, and biochemical methods to analyze the effect of GTP on EB1 in vitro. Isothermal titration calorimetry and tryptophan fluorescence quenching experiments demonstrated that EB1 binds to GTP with a dissociation constant ~30 μM. Circular dichroism measurements showed that EB1 undergoes changes in its secondary structure on binding GTP. Size-exclusion chromatography and urea-induced unfolding analyses revealed that GTP binding induces dissociation of the EB1 dimer to monomers. Size-exclusion chromatography followed by biochemical analysis further determined that EB1-GTP binding involves association of approximately one molecule of GTP per EB1 monomer. The results reveal a hitherto unknown GTP-dependent mechanism of dimer-to-monomer transition in EB1 and further implicate its possible role in regulating the stability of the EB1 dimer vs monomer as well as plus-end regulation in cells.

EB1 regulates attachment of Ska1 with microtubules by forming extended structures on the microtubule lattice

“…The plasmids containing spc34 and dad1 with 6xHis-tag were expressed under induction of arabinose 8 . All the His-tagged and GST-tagged proteins were expressed in Escherichia coli BL21-DE3 cells and purified by Ni 2+ -NTA (QIAGEN) and glutathione Sepharose (GE Healthcare), respectively 56 . Tubulin was purified from goat brain by repetitive cycles of polymerization and depolymerization 56 .…”

“…All the His-tagged and GST-tagged proteins were expressed in Escherichia coli BL21-DE3 cells and purified by Ni 2+ -NTA (QIAGEN) and glutathione Sepharose (GE Healthcare), respectively 56 . Tubulin was purified from goat brain by repetitive cycles of polymerization and depolymerization 56 . Protein concentrations were estimated using the Bradford method using BSA as standard 57 .…”

Signaling Scaffold Protein IQGAP1 Interacts with Microtubule Plus-end Tracking Protein SKAP and Links Dynamic Microtubule Plus-end to Steer Cell Migration

“…The crystal structure of the EB1 COOH-terminal domain reveals a novel homodimeric-fold comprised of a coiled coil and a four-helix bundle motif (12). A recent study reported a GTP-dependent mechanism of dimer-to-monomer transition in EB1 (15). Moreover, ϩTIPs functioning is controlled by conformational modifications.…”

The drebrin/EB3 pathway drives invasive activity in prostate cancer

“…EB1 recognizes GTP-bound tubulin in the GTP-cap region at the plus-end of microtubules. 42 The more proximal binding of EB3 is consistent with EB3 recognizing GDP-bound tubulin. The differential localization of these two +TIP proteins on the same microtubule has implications for the consequences of drebrin binding to EB3.…”