Structural determinants for EB1-mediated recruitment of APC and spectraplakins to the microtubule plus end

Abstract

EB1 is a member of a conserved protein family that localizes to growing microtubule plus ends. EB1 proteins also recruit cell polarity and signaling molecules to microtubule tips. However, the mechanism by which EB1 recognizes cargo is unknown. Here, we have defined a repeat sequence in adenomatous polyposis coli (APC) that binds to EB1's COOH-terminal domain and identified a similar sequence in members of the microtubule actin cross-linking factor (MACF) family of spectraplakins. We show that MACFs directly bind EB1 and exhibit EB1-dependent plus end tracking in vivo. To understand how EB1 recognizes APC and MACFs, we solved the crystal structure of the EB1 COOH-terminal domain. The structure reveals a novel homodimeric fold comprised of a coiled coil and four-helix bundle motif. Mutational analysis reveals that the cargo binding site for MACFs maps to a cluster of conserved residues at the junction between the coiled coil and four-helix bundle. These results provide a structural understanding of how EB1 binds two regulators of microtubule-based cell polarity.

Adenomatous polyposis coli plays a key role, in vivo, in coordinating assembly of the neuronal nicotinic postsynaptic complex

“…Thus, APC positions EB1-tagged microtubules to precise positions of the cell surface. MACF directly binds to EB1, microtubules and F-actin, and is required for tethering EB1-tagged microtubules to the submembranous F-actin network at specialized junctions (Kodama et al, 2003;Subramanian et al, 2003;Slep et al, 2005). Microtubule binding to all three proteins, APC, EB1 and MACF, promotes their stabilization and leads to more frequent and longer lasting attachments (Dikovskaya et al, 2001;Tirnauer et al, 2002;Kodama et al, 2003;Reilein and Nelson, 2005).…”

“…1E). Our model is further based on studies in non-neuronal cells that show IQGAP1 and macrophin (MACF, microtubule-actin cross-linking factor) interact with APC and EB1 Slep et al, 2005). These proteins bind to and regulate the dynamics of EB1-tagged microtubules and submembranous F-actin at specialized intercellular junctions (Barth et al, 2002;Jefferson et al, 2004;Noritake et al, 2004).…”

Lipid transfer protein binding of unmodified natural lipids as assessed by surface plasmon resonance methodology

“…Only one other study is known in which a biotinylated AviTag-like extension was used to immobilize a protein for SPR analysis [48]. However, this involved a protein-protein interaction system for which only qualitative effects of mutagenesis (not kinetic and equilibrium constants) were described.…”

Functional and Genetic Analysis of Spectraplakins in Drosophila

“…Nevertheless, S2 cells are a powerful tool to unravel new molecular mechanisms which can then be studied in other contexts. In this way, S2 cells have provided us with first descriptions of mechanisms underlying the recruitment of Shot to MT plus ends (Applewhite et al, 2010;Slep et al, 2005) and a novel intramolecular autoinhibition mechanism (Applewhite et al, 2013).…”