XTACC3–XMAP215 association reveals an asymmetric interaction promoting microtubule elongation

Abstract

chTOG is a conserved microtubule polymerase that catalyses the addition of tubulin dimers to promote microtubule growth. chTOG interacts with TACC3, a member of the transforming acidic coiled-coil (TACC) family. Here we analyse their association using the Xenopus homologues, XTACC3 (TACC3) and XMAP215 (chTOG), dissecting the mechanism by which their interaction promotes microtubule elongation during spindle assembly. Using SAXS, we show that the TACC domain (TD) is an elongated structure that mediates the interaction with the C terminus of XMAP215. Our data suggest that one TD and two XMAP215 molecules associate to form a four-helix coiled-coil complex. A hybrid methods approach was used to define the precise regions of the TACC heptad repeat and the XMAP215 C terminus required for assembly and functioning of the complex. We show that XTACC3 can induce the recruitment of larger amounts of XMAP215 by increasing its local concentration, thereby promoting efficient microtubule elongation during mitosis.

Structural insight into the XTACC3/XMAP215 interaction from CD and NMR studies on model peptides

“…On the other hand, the C‐terminal binding region of XMAP is predicted to be a coiled‐coil. When isolated, it tends to populate helical structures and a short peptide XMAP‐pCt (Table ), with similar structural propensities was described . It has also been shown that the pCt peptide has a good affinity to TD4 and, for this reason, it is an appropriate model to mimic the intermolecular interactions of the full‐length protein.…”

“…Mortuza et al, defined a minimal active binding TACC domain (TD4, 26 kDa) for the protein XTACC3 (the Xenopus homologue of the human TACC3, 81% sequence similarity) as well as that of XMAP215 (Ct, 7kDa). It is well known that human centrosomal proteins show a significant tendency to adopt both unstructured and coiled‐coils conformers which complicates the determination of their structure at atomic resolution.…”

“…However, a medium resolution 3D model is available based on small‐angle X‐ray scattering (SAXS) data. It is an elongated helical structure, where the biologically relevant helix α2 and the C‐terminus of helix α4 face each other in an antiparallel orientation …”

“…Based on biophysical and biological data, a structural model for the XTACC3‐XMAP215 (1:2 complex) interaction was proposed . In this model (Figure ), the helical structure of XMAP‐pCt lies in the grooves between helix α2 and the C‐terminus of helix α4.…”

From meiosis to mitosis – the sperm centrosome defines the kinetics of spindle assembly after fertilization in Xenopus

“…We and others have previously shown that XTACC3 promotes microtubule assembly at the centrosome in Xenopus egg extracts. Indeed, depletion of XTACC3 results in a significant reduction of microtubules by 40% in the centrosome asters, but it does not impair bipolar spindle formation in cycled egg extracts (Kinoshita et al, 2005;Mortuza et al, 2014;Peset et al, 2005;Sardon et al, 2008). We first incubated Xenopus sperm nuclei carrying one centrosome in mock-(control) or XTACC3-depleted CSF extracts ( Fig.…”