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.
Keywords: CAP-Gly; CLIP-170; EB1; SXIP; cytoskeleton; isothermal titration calorimetry (ITC); microtubule; microtubule-associated protein (MAP); protein–protein interaction; tubulin.
© 2019 Chen et al.