Insights into EB structure and the role of its C-terminal domain in discriminating microtubule tips from lattice

Biomolecular Research, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland.
Molecular biology of the cell (Impact Factor: 4.47). 08/2011; 22(16):2912-23. DOI: 10.1091/mbc.E11-01-0017
Source: PubMed


End-binding proteins (EBs) comprise a conserved family of microtubule plus end-tracking proteins. The concerted action of calponin homology (CH), linker, and C-terminal domains of EBs is important for their autonomous microtubule tip tracking, regulation of microtubule dynamics, and recruitment of numerous partners to microtubule ends. Here we report the detailed structural and biochemical analysis of mammalian EBs. Small-angle X-ray scattering, electron microscopy, and chemical cross-linking in combination with mass spectrometry indicate that EBs are elongated molecules with two interacting CH domains, an arrangement reminiscent of that seen in other microtubule- and actin-binding proteins. Removal of the negatively charged C-terminal tail did not affect the overall conformation of EBs; however, it increased the dwell times of EBs on the microtubule lattice in microtubule tip-tracking reconstitution experiments. An even more stable association with the microtubule lattice was observed when the entire negatively charged C-terminal domain of EBs was replaced by a neutral coiled-coil motif. In contrast, the interaction of EBs with growing microtubule tips was not significantly affected by these C-terminal domain mutations. Our data indicate that long-range electrostatic repulsive interactions between the C-terminus and the microtubule lattice drive the specificity of EBs for growing microtubule ends.

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Available from: Ihor Smal, Nov 07, 2014
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    • "Repulsive forces between the negatively charged C-terminus and the microtubule lattice are also likely to contribute to the plusend localisation (Buey et al., 2011). EB1 and EB3, but to a lesser extent EB2, interact via the C-terminus with most other +TIPs including APC (adenomatous polyposis coli), CLIPs (cytoplasmic linker proteins), CLASPs (CLIP-associated proteins) and the microtubule-actin cross-linking spectraplakin ACF7 (MACF1). "
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    ABSTRACT: Microtubule end-binding (EB) proteins influence microtubule dynamic instability, a process essential for microtubule reorganisation during apico-basal epithelial differentiation. Here we establish for the first time that EB2, but not EB1, expression is critical for initial microtubule reorganisation during apico-basal epithelial differentiation, and that EB2 downregulation promotes bundle formation. EB2 siRNA knockdown during early stages of apico-basal differentiation prevented microtubule reorganisation, while its downregulation at later stages promoted microtubule stability and bundle formation. Interestingly, while EB1 is not essential for microtubule reorganisation its knockdown prevented apico-basal bundle formation and epithelial elongation. EB2 siRNA depletion in undifferentiated epithelial cells induced formation of straight, less dynamic microtubules with EB1 and ACF7 lattice association and co-alignment with actin filaments, a phenotype that could be rescued by formin inhibition. Importantly, in situ inner ear and intestinal crypt epithelial tissue revealed direct correlations between low level of EB2 expression and presence of apico-basal microtubule bundles, which were absent where EB2 was elevated. EB2 is evidently important for initial microtubule reorganisation during epithelial polarisation, while its downregulation facilitates EB1/ACF7 microtubule lattice association, microtubule-actin filament co-alignment and bundle formation. The spatiotemporal expression of EB2 thus dramatically influences microtubule organisation, EB1/ACF7 deployment and epithelial differentiation.
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    • "The K d values obtained for the EB1–ClipCG2 and EB1– p150CG[A49M] complexes are very similar to the ones obtained for the interactions between the isolated C-terminal domain of EB1 (denoted EB1c) and both the CAP-Gly domains (Weisbrich et al., 2007; Honnappa et al., 2006), demonstrating that the N-terminal moiety of EB1 does not influence the binding reaction. This finding is in agreement with recent small angle X-ray scattering data suggesting that mammalian EBs adopt an elongated conformation in solution with no detectable interactions between the N-and C-terminal domains (Buey et al., 2011). The similar affinities measured for the EB3 homodimer and the EB13 heterodimer suggests that, at least with respect to CLIP-170 and p150 glued CAP-Gly binding, the two EB dimer versions are indistinguishable. "
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    ABSTRACT: End binding proteins (EBs) track growing microtubule ends and play a master role in organizing dynamic protein networks. Mammalian cells express up to three different EBs (EB1, EB2, and EB3). Besides forming homodimers, EB1 and EB3 also assemble into heterodimers. One group of EB-binding partners encompasses proteins that harbor CAP-Gly domains. The binding properties of the different EBs towards CAP-Gly proteins have not been systematically investigated. This information is, however, important to compare and contrast functional differences. Here we analyzed the interactions between CLIP-170 and p150(glued) CAP-Gly domains with the three EB homodimers and the EB1-EB3 heterodimer. Using isothermal titration calorimetry we observed that some EBs bind to the individual CAP-Gly domains with similar affinities while others interact with their targets with pronounced differences. We further found that the two types of CAP-Gly domains use alternative mechanisms to target the C-terminal domains of EBs. We succeeded to solve the crystal structure of a complex composed of a heterodimer of EB1 and EB3 C-termini together with the CAP-Gly domain of p150(glued). Together, our results provide mechanistic insights into the interaction properties of EBs and offer a molecular framework for the systematic investigation of their functional differences in cells.
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