Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR |
| |
Authors: | Teppei Kanaba Ryoko Maesaki Tomoyuki Mori Yutaka Ito Toshio Hakoshima Masaki Mishima |
| |
Institution: | 1. Graduate School of Science and Engineering, Tokyo Metropolitan University, 1-1 Minamiosawa Hachioji 192-0397, Japan;2. Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, Japan |
| |
Abstract: | End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p (Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTPγS-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371–82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTPγS-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371–82). Notably, the N-terminal tip of helix α6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of β-tubulin in GTPγS-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein. |
| |
Keywords: | HSQC heteronuclear single quantum correlation spectroscopy TROSY Transverse relaxation optimized spectroscopy CSP chemical shift perturbation TCS transferred cross-saturation + TIPs plus-end tracking proteins MT microtubule EB1 end-binding protein 1 CH domain calponin homology domain CAP-Gly domain cytoskeleton-associated protein glycine-rich domain CLIP cytoplasmic linker protein APC adenomatous polyposis coli |
本文献已被 ScienceDirect 等数据库收录! |
|