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J Biol Chem. 2019 Jan 18;294(3):918-931. doi: 10.1074/jbc.RA118.006125. Epub 2018 Nov 19.

Mapping multivalency in the CLIP-170-EB1 microtubule plus-end complex.

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From the Key Laboratory of Resources Biology and Biotechnology in Western China, Ministry of Education, College of Life Sciences, Northwest University, Xi'an, Shaanxi 710069, China.
the Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, and.
the Department of Neurology, University of North Carolina, Chapel Hill, North Carolina 27599.
the Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599-3280, and


Cytoplasmic linker protein 170 (CLIP-170) is a microtubule plus-end factor that links vesicles to microtubules and recruits the dynein-dynactin complex to microtubule plus ends. CLIP-170 plus-end localization is end binding 1 (EB1)-dependent. CLIP-170 contains two N-terminal cytoskeleton-associated protein glycine-rich (CAP-Gly) domains flanked by serine-rich regions. The CAP-Gly domains are known EB1-binding domains, and the serine-rich regions have also been implicated in CLIP-170's microtubule plus-end localization mechanism. However, the determinants in these serine-rich regions have not been identified. Here we elucidated multiple EB1-binding modules in the CLIP-170 N-terminal region. Using isothermal titration calorimetry and size-exclusion chromatography, we mapped and biophysically characterized these EB1-binding modules, including the two CAP-Gly domains, a bridging SXIP motif, and a unique array of divergent SXIP-like motifs located N-terminally to the first CAP-Gly domain. We found that, unlike the EB1-binding mode of the CAP-Gly domain in the dynactin-associated protein p150Glued, which dually engages the EB1 C-terminal EEY motif as well as the EB homology domain and sterically occludes SXIP motif binding, the CLIP-170 CAP-Gly domains engage only the EEY motif, enabling the flanking SXIP and SXIP-like motifs to bind the EB homology domain. These multivalent EB1-binding modules provided avidity to the CLIP-170-EB1 interaction, likely clarifying why CLIP-170 preferentially binds EB1 rather than the α-tubulin C-terminal EEY motif. Our finding that CLIP-170 has multiple non-CAP-Gly EB1-binding modules may explain why autoinhibition of CLIP-170 GAP-Gly domains does not fully abrogate its microtubule plus-end localization. This work expands our understanding of EB1-binding motifs and their multivalent networks.


CAP-Gly; CLIP-170; EB1; SXIP; cytoskeleton; isothermal titration calorimetry (ITC); microtubule; microtubule-associated protein (MAP); protein–protein interaction; tubulin

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