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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    • "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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