Structural basis of tubulin recruitment and assembly by microtubule polymerases with tumor overexpressed gene (TOG) domain arrays.

Stanley Nithianantham,Jawdat Al-Bassam,Brian D Cook,Fred Chang,Madeleine Beans,Fei Guo

doi:10.7554/elife.38922

Stanley Nithianantham, Jawdat Al-Bassam + Show 4 more

Open Access

https://doi.org/10.7554/elife.38922

Copy DOI

Abstract

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of tumor overexpressed gene (TOG) domains, yet their mechanism remains unknown. Here, we describe the biochemical and structural basis for TOG arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 subunits, in which each domain exhibits a distinct exchange rate for tubulin. X-ray structures revealed square-shaped assemblies composed of pseudo-dimeric TOG1-TOG2 subunits assembled head-to-tail, positioning four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking and electron microscopy show Alp14-tubulin forms square assemblies in solution, and inactivating their interfaces destabilize this organization without influencing tubulin binding. An X-ray structure determined using approach to modulate tubulin polymerization revealed an unfurled assembly, in which TOG1-TOG2 uniquely bind to two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies that then unfurl, facilitating their concerted polymerization into protofilaments.

Highlights

Microtubules (MTs) are highly dynamic polarized polymers that perform critical and diverse cellular functions including formation of bipolar mitotic spindles, intracellular organization, and modulation of cell development and cell migration (Akhmanova and Steinmetz, 2008; Akhmanova and Steinmetz, 2015)
We studied the ab-tubulin binding capacities and stoichiometries of near native monomeric and dimeric Alp14, which both consist of TOG1-TOG2 arrays and differ by the presence of a C-terminal SK-rich region and dimerization coiled-coil domain
Using quantitative size-exclusion chromatography (SEC) with multi-angle light scattering (SEC-MALS), we measured the ab-tubulin binding stoichiometry for monomeric Alp14, and dimeric Alp14 at 80–100 mM KCl ionic strength (Figure 1A–B, Figure 1D; details described in Figure 1—figure supplement 1A–C,D–F; SEC-MALS control experiments shown in Figure 1—figure supplement 2G–H; Table 1) (Al-Bassam et al, 2012)

Summary

Introduction

Microtubules (MTs) are highly dynamic polarized polymers that perform critical and diverse cellular functions including formation of bipolar mitotic spindles, intracellular organization, and modulation of cell development and cell migration (Akhmanova and Steinmetz, 2008; Akhmanova and Steinmetz, 2015). Polymerization of ab-tubulin and GTP hydrolysis are regulated by conserved proteins that bind at MT plus-ends or along MT lattices (Akhmanova and Steinmetz, 2008; Akhmanova and Steinmetz, 2011; Akhmanova and Steinmetz, 2015; AlBassam and Chang, 2011; Al-Bassam et al, 2010; Brouhard and Rice, 2014). The XMAP215/Stu2/ Alp MT polymerases are among the best-studied families of MT regulators They localize to the extreme tips of MT plus-ends and accelerate ab-tubulin polymerization in eukaryotes (Akhmanova and Steinmetz, 2011; Akhmanova and Steinmetz, 2015; Al-Bassam and Chang, 2011; Maurer et al, 2014).

Objectives

Results

Conclusion