Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1242/jcs.127845

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1107/S1744309112045551

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1074/jbc.M112.373803

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1016/j.yexcr.2011.11.006

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Language：English   Publishing type：Research paper (scientific journal)  

XH/pi hydrogen bonds have been predicted to make important contributions to protein structure and function. NMR evidence is presented for an OH/pi interaction between a highly conserved threonine and phenylalanine pair found specifically in CAP-Gly domains associated with mictrotubule plus ends. The functional contribution of this nonclassical hydrogen bond in target peptide recognition is demonstrated via subtle point mutagenesis. The OH/pi interaction is part of a TxFxxxxW motif that comprises a conserved "threonine clasp" that defines function in CAP-Gly domains.

DOI： 10.1021/ja805576n

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

CLIP170 and p150(Glued) localize to the plus ends of growing microtubules. Using crystallography and NMR, we show that autoinhibitory interactions within CLIP170 use the same binding determinants as CLIP170's intermolecular interactions with p150(Glued). These interactions have both similar and distinct features when compared with the p150(Glued)-EB1 complex. Our data thus demonstrate that regulation of microtubule dynamics by plus end-tracking proteins (+TIPs) occurs through direct competition between homologous binding interfaces.

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Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

DOI： 10.1016/j.jmb.2007.06.037

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued. Each molecule of the EB1 dimer contains two helices forming a conserved four-helix bundle, while also providing p150Glued binding sites in its flexible tail region. Combining crystallography, NMR, and mutational analyses, our studies reveal the critical interacting elements of both EB1 and p150Glued, whose mutation alters microtubule polymerization activity. Moreover, removal of the key flexible tail from EB1 activates microtubule assembly by EB1 alone, suggesting that the flexible tail negatively regulates EB1 activity. We, therefore, propose that EB1 possesses an auto-inhibited conformation, which is relieved by p150Glued as an allosteric activator.

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Language：Japanese   Publishing type：Research paper (scientific journal)  

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

The end-binding protein 1 (EB1) family is a highly conserved group of proteins that localizes to the plus-ends of microtubules. EB1 has been shown to play an important role in regulating microtubule dynamics and chromosome segregation, but its regulation mechanism is poorly understood. We have determined the 1.45-A resolution crystal structure of the amino-terminal domain of EB1, which is essential for microtubule binding, and show that it forms a calponin homology (CH) domain fold that is found in many proteins involved in the actin cytoskeleton. The functional CH domain for actin binding is a tandem pair, whereas EB1 is the first example of a single CH domain that can associate with the microtubule filament. Although our biochemical study shows that microtubule binding of EB1 is electrostatic in part, our mutational analysis suggests that the hydrophobic network, which is partially exposed in our crystal structure, is also important for the association. We propose that, like other actin-binding CH domains, EB1 employs the hydrophobic interaction to bind to microtubules.

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