title: Molecular insights into mammalian end-binding protein heterodimerization.
abstract: Microtubule plus-end tracking proteins (+TIPs) are involved in many microtubule-based processes. End binding (EB) proteins constitute a highly conserved family of +TIPs. They play a pivotal role in regulating microtubule dynamics and in the recruitment of diverse +TIPs to growing microtubule plus ends. Here we used a combination of methods to investigate the dimerization properties of the three human EB proteins EB1, EB2, and EB3. Based on Förster resonance energy transfer, we demonstrate that the C-terminal dimerization domains of EBs (EBc) can readily exchange their chains in solution. We further document that EB1c and EB3c preferentially form heterodimers, whereas EB2c does not participate significantly in the formation of heterotypic complexes. Measurements of the reaction thermodynamics and kinetics, homology modeling, and mutagenesis provide details of the molecular determinants of homo- versus heterodimer formation of EBc domains. Fluorescence spectroscopy and nuclear magnetic resonance studies in the presence of the cytoskeleton-associated protein-glycine-rich domains of either CLIP-170 or p150(glued) or of a fragment derived from the adenomatous polyposis coli tumor suppressor protein show that chain exchange of EBc domains can be controlled by binding partners. Extension of these studies of the EBc domains to full-length EBs demonstrate that heterodimer formation between EB1 and EB3, but not between EB2 and the other two EBs, occurs both in vitro and in cells as revealed by live cell imaging. Together, our data provide molecular insights for rationalizing the dominant negative control by C-terminal EB domains and form a basis for understanding the functional role of heterotypic chain exchange by EBs in cells.
authors.affiliation: Biomolecular Research, Structural Biology, the Paul Scherrer Institut, CH-5232 Villigen PSI, Switzerland.
authors.forename: Christian O
authors.initials: CO
authors.lastname: De Groot
authors.forename: Ilian
authors.initials: I
authors.lastname: Jelesarov
authors.forename: Fred F
authors.initials: FF
authors.lastname: Damberger
authors.forename: Sasa
authors.initials: S
authors.lastname: Bjelić
authors.forename: Martin A
authors.initials: MA
authors.lastname: Schärer
authors.forename: Neel S
authors.initials: NS
authors.lastname: Bhavesh
authors.forename: Ilia
authors.initials: I
authors.lastname: Grigoriev
authors.forename: Ruben M
authors.initials: RM
authors.lastname: Buey
authors.forename: Kurt
authors.initials: K
authors.lastname: Wüthrich
authors.forename: Guido
authors.initials: G
authors.lastname: Capitani
authors.forename: Anna
authors.initials: A
authors.lastname: Akhmanova
authors.forename: Michel O
authors.initials: MO
authors.lastname: Steinmetz
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000072159:Dynactin Complex; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization / Q000502:physiology*; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: C000606360:DCTN1 protein, human; D000072159:Dynactin Complex; C500985:MAPRE1 protein, human; C403664:MAPRE3 protein, human; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D011994:Recombinant Proteins; C076744:cytoplasmic linker protein 170