Crystal Structure of the Amino-terminal Microtubule-binding Domain of End-binding Protein 1 (EB1)

Ikuko Hayashi,Mitsuhiko Ikura

doi:10.1074/jbc.m305773200

Abstract

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.

Highlights

Microtubules (MTs)1 are an essential component of the cytoskeleton, underlying the fundamental processes of cell morphogenesis, cell motility, and cell division
We have determined the 1.45-Å resolution crystal structure of the amino-terminal domain of end-binding protein 1 (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
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 important for the association

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—The human EB1 amino-terminal MT-binding domain (residues 1–130) was generated by PCR from a human adult brain cDNA library and cloned into pET15b vector (Novagen) using NdeI and XhoI restriction enzyme sites. The eluted protein was digested with thrombin protease (Sigma) to remove the His tag and purified by gel filtration (Superdex 75, Amersham Biosciences) with a buffer containing 10 mM Tris, pH 8.0, 0.1 M NaCl and 1 mM dithiothreitol. En mutants were expressed in E. coli cells and purified as described for the wild type. The crystals of the P21 space group were grown over a reservoir with 20% polyethylene glycol 3350, 0.2 M ammonium sulfate, and 0.1 M MES, pH 6.0. Another crystal form (P43212) was grown over a reservoir containing 3 M ammonium sulfate and 0.1 M sodium citrate, pH 5.5.

Protein Data Bank accession code

RESULTS AND DISCUSSION

Ikuko Hayashi and Mitsuhiko Ikura