title: Insights into EB1 structure and the role of its C-terminal domain for discriminating microtubule tips from the lattice.
abstract: 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.
authors: Biomolecular Research, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland. ruben.martinez@usal.es
authors: Rubén M
authors: RM
authors: Buey
authors: Renu
authors: R
authors: Mohan
authors: Kris
authors: K
authors: Leslie
authors: Thomas
authors: T
authors: Walzthoeni
authors: John H
authors: JH
authors: Missimer
authors: Andreas
authors: A
authors: Menzel
authors: Saša
authors: S
authors: Bjelic
authors: Katja
authors: K
authors: Bargsten
authors: Ilya
authors: I
authors: Grigoriev
authors: Ihor
authors: I
authors: Smal
authors: Erik
authors: E
authors: Meijering
authors: Ruedi
authors: R
authors: Aebersold
authors: Anna
authors: A
authors: Akhmanova
authors: Michel O
authors: MO
authors: Steinmetz
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry; D036681:Fluorescence Recovery After Photobleaching; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry; D011485:Protein Binding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D014961:X-Ray Diffraction
chemical_list: D003432:Cross-Linking Reagents; C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins; D010446:Peptide Fragments; D008239:Lysine