Structure-function analysis of how XMAP215 binds γ-Tubulin for microtubule nucleation

Abstract

Microtubules are long, cylindrical polymers that are crucial to cell structure, function, and division. Until recently, γ-Tubulin Ring Complex (γ-TuRC) was recognized as the primary microtubule nucleating protein. However, recent studies have revealed that a previously known microtubule polymerase, XMAP215, synergistically nucleates microtubules with γ-TuRC and binds γ-tubulin via its C-terminal domain. Yet, the exact binding region of this interaction remains to be determined. XMAP215 is a multi-domain protein containing five TOG (Tumor Overexpressed Gene) domains that bind and recruit αβ-tubulins during its polymerase activity. However, the discovery of a cryptic sixth TOG domain within the highly conserved C-terminus of XMAP215 has led to the hypothesis that this TOG6 domain may serve as the primary γ-tubulin binding region in XMAP215 by mirroring the tubulin binding abilities of classical TOGs. Various truncation constructs of the C-terminal domain of XMAP215 were expressed and purified to further elucidate the exact binding interface within XMAP215’s C-terminal region. These constructs were tested in pull-down binding assays with γ-tubulin to assess the individual binding abilities of the Linker-4, TOG5, C-terminus, and TOG6 domains. I conclude that the C-terminus of XMAP215 is sufficient and necessary for γ-tubulin binding in XMAP215, whereas the Linker-4 and TOG5 domains are not required for this binding interaction. Though the TOG6 domain was shown to bind γ-tubulin, its binding strength was not equivalent to that of the full-length C-terminus. γ-tubulin binding also occurred beyond the TOG6 domain, suggesting that the binding schema within XMAP215’s C-terminus is more complex than the initial hypothesis. Still, the findings of this investigation further solidify the synergistic nucleating function of XMAP215 by identifying a structural basis for its binding interaction with γ-tubulin.