The XMAP215 family drives microtubule polymerization using a structurally diverse TOG array

Campbell, Jaime Nicole

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March 20, 2019

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Campbell, Jaime Nicole
- Affiliation: School of Medicine, Department of Biochemistry and Biophysics

Abstract

XMAP215 family members are potent microtubule (MT) polymerases, with mutants displaying reduced MT growth rates and aberrant spindle morphologies. XMAP215 proteins contain arrayed TOG domains that bind tubulin. Whether these TOG domains are functionally equivalent and how they collectively operate to drive polymerization remains unknown. Here, we present crystal structures of TOG4 from Drosophila Msps and human ch-TOG. These TOG4 structures architecturally depart from the structures of TOG domains 1 and 2, revealing a conserved domain bend that predicts a novel engagement with α-tubulin. In vitro assays show differential tubulin-binding affinities across the TOG array, as well as differential effects on MT nucleation and polymerization. We used Drosophila S2 cells depleted of endogenous Msps to assess the importance of individual TOG domains. While a TOG1-4 array largely rescues MT polymerization rates, mutating tubulin-binding determinants in any single TOG domain dramatically reduces rescue activity. Our work highlights the structurally diverse, yet positionally conserved TOG array that drives MT polymerization. XMAP215 family function in mitosis has been closely linked to a centrosomal binding factor, the TACC family of proteins. TACC family proteins have proven important structural components of the mitotic spindle apparatus. Disruption of TACC function causes disorganized and unstable spindle microtubules leading to multiple biological consequences including chromosome instability, developmental problems, and cancer (reviewed in Thakur et al., 2013). The mechanism in which TACC family proteins functions at the centrosome is still poorly understood. Our study aims to further the field of centrosome biology by understanding how members of the TACC family are interacting with its binding partners, specifically XMAP215 family members. Here we have identified a minimal domain of the Drosophila TACC family member, DTACC, that confers TACC dimerization, localizes to spindle poles in mitosis and tracks along the MT plus-end potentially through an interaction with the XMAP215 family member Msps in interphase. Mutational analysis has identified specific residues within this minimal domain important for Msps binding and to centrosome localization. Further analysis to structurally characterize the DTACC-Msps interaction is ongoing in the lab.

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