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-glycinerich 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.

EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms

“…7H,J and data not shown). Since C-terminal EB1 and EB3 fragments both interfere with the formation of EB3 dimers (De Groot et al, 2010;Komarova et al, 2009), these results reinforce the notion that EB3 affects the formation of rootlet filaments. Rootletin interacts with CEP250 and -catenin, which are involved in regulating centrosome cohesion through a complex mechanism that involves Nek2, Axin2 (also known as conductin) and Wnt signaling (Bahe et al, 2005;Bahmanyar et al, 2008;Hadjihannas et al, 2010).…”

“…S2C). Since EB1 and EB3 form homodimers and also heterodimerize with each other but not with EB2 (De Groot et al, 2010;Komarova et al, 2009), GFP-EB1-C and GFP-EB3-C are expected to disrupt native EB1-EB3 homoand heterodimers, but not EB2 homodimers, whereas GFP-EB2-C is expected to affect the EB2 pool only. The above results are, therefore, consistent with the siRNA results ( Fig.…”

“…Mammals contain three EBs, which are similar in structure (EB1, EB2, and EB3; 57-66% amino acid identity) and are encoded by separate genes (Juwana et al, 1999;Su and Qi, 2001). All three EBs exist as homodimers in vivo and in vitro, and EB1 and EB3 also form a heterodimeric complex that might be functionally distinct from the homodimers (De Groot et al, 2010;Komarova et al, 2009). EBs contain an Nterminal MT-binding calponin homology (CH) domain, a flexible linker and a C-terminal coiled-coil domain region that mediates EB dimerization and partially overlaps with a unique EB homology domain (Akhmanova and Steinmetz, 2008;De Groot et al, 2010;Hayashi and Ikura, 2003;Honnappa et al, 2005;Komarova et al, 2009;Slep et al, 2005).…”

“…All three EBs exist as homodimers in vivo and in vitro, and EB1 and EB3 also form a heterodimeric complex that might be functionally distinct from the homodimers (De Groot et al, 2010;Komarova et al, 2009). EBs contain an Nterminal MT-binding calponin homology (CH) domain, a flexible linker and a C-terminal coiled-coil domain region that mediates EB dimerization and partially overlaps with a unique EB homology domain (Akhmanova and Steinmetz, 2008;De Groot et al, 2010;Hayashi and Ikura, 2003;Honnappa et al, 2005;Komarova et al, 2009;Slep et al, 2005). The EB homology domain is followed by a tail of 20-30 amino acid residues that frequently ends with the sequence EEY/F (Honnappa et al, 2005;Slep et al, 2005).…”

Interaction of mammalian end binding proteins with CAP-Gly domains of CLIP-170 and p150glued

“…Despite high sequence identity between EB1 and EB3, the formation of the EB1-EB3 heterodimer is energetically favorable (De Groot et al, 2010). EBs are relatively small dimeric proteins that contain three distinct domains: a conserved N-terminal calponin homology (CH) domain important for microtubule binding ($130 residues; Hayashi and Ikura, 2003;Komarova et al, 2009;Slep and Vale, 2007), a variable polypeptide linker ($70 residues), and a conserved C-terminal dimerization domain ($80 residues; Honnappa et al, 2005Honnappa et al, , 2009Slep et al, 2005;De Groot et al, 2010). The Cterminal domain (denoted EBc) comprises the unique EB homology (EBH) domain and a disordered C-terminal tail.…”