GTP-binding facilitates EB1 recruitment onto microtubules by relieving its auto-inhibition

Abstract

Microtubule plus end-binding protein, EB1 is a key regulator of microtubule dynamics. Auto-inhibitory interaction in EB1 has previously been shown to inhibit its ability to bind to microtubules and regulate microtubule dynamics. However, the factors that promote its microtubule regulatory activity by over-coming the auto-inhibition are less known. Here, we show that GTP plays a critical role in promoting the microtubule-targeting activity of EB1 by suppressing its auto-inhibition. Our biophysical data demonstrate that GTP binds to EB1 at a distinct site in its conserved N-terminal domain. Detailed analyses reveal that GTP-binding suppresses the intra-molecular inhibitory interaction between the globular N-terminus and the C-terminal coiled-coil domain. We further show that mutation of the GTP-binding site residues in N-terminus weakens the affinity for GTP, but also for the C-terminus, indicating overlapping binding sites. Confocal imaging and biochemical analysis reveal that EB1 localization on the microtubules is significantly increased upon mutations of the GTP-binding site residues. The results demonstrate a unique role of GTP in facilitating EB1 interaction with the microtubules by relieving its intra-molecular inhibition. They also implicate that GTP-binding may regulate the functions of EB1 on the cellular microtubules.

Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules

“…Even in instances of Ndc80 overexpression in yeast or mammalian cells, excess Ndc80 is not found along the microtubule lattice in the mitotic spindle (Tang and Toda, 2015; Diaz-Rodríguez et al, 2008). This specific localization to the tips of microtubules mirrors the localization of +TIPs, which often require multiple modes of regulation to ensure accurate targeting to the microtubule plus-end as well as appropriate function (Gireesh et al, 2018). Thus, there are likely multiple regulatory mechanisms to ensure both proper localization and function of the Ndc80 complex in cells.…”

Validation and classification of RNA binding proteins identified by mRNA interactome capture

“…Thus, the binding of EB1 to RNA which we observed in vitro might merely demonstrate electrostatic interaction with the negatively charged sugar backbone of the RNA molecule, or alternatively with its individual building blocks -the nucleotides. It was recently shown that EB1 exhibits direct binding to GTP (Gireesh et al 2018) using the same surface it uses for binding to MTs and RNA.…”

Validation and classification of RNA binding proteins identified by mRNA interactome capture

“…Thus, the binding of EB1 to RNA which we observed in vitro might merely demonstrate electrostatic interaction with the negatively charged sugar backbone of the RNA molecule, or alternatively with its individual building blocks – the nucleotides. It was recently shown that EB1 exhibits direct binding to GTP (Gireesh et al, 2018) using the same surface it uses for binding to MTs and RNA. Other studies have also demonstrated that small GTPases or nucleotide binding enzymes are enriched in the interactome of certain RNAs (Castello et al, 2015; Liu et al, 2019).…”