Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR

Kanaba, Teppei; Maesaki, Ryoko; Mori, Tomoyuki; Ito, Yutaka; Hakoshima, Toshio; Mishima, Masaki

doi:10.1016/j.bbapap.2012.10.013

Cited by 11 publications

(16 citation statements)

References 27 publications

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“…Previous studies have shown that EB1 C-terminus negatively regulates the microtubule binding- and assembly-promoting activity of EB1 by binding to the CH domain and countering the CH domain–microtubule interaction 7 , 8 . Consistent with these findings, another study has shown that the residues of the CH domain involved in the interaction between CH domain and EB1 C-terminus, largely overlap with the microtubule-binding sites of the CH domain 26 . However, the mechanism how EB1 is recruited to the microtubules by overcoming this intra-molecular inhibition remains poorly understood.…”

Section: Introductionsupporting

confidence: 56%

“…We next characterized the binding sites of GTP in EB1n by NMR. First, the NMR backbone resonance re-assignment 26 was carried out using standard triple resonance NMR experiments using double labeled 13 C- 15 N EB1n protein. We could identify around 90% of the backbone resonances of EB1n.…”

Section: Resultsmentioning

confidence: 99%

“…The N-terminus largely consists of a conserved calponin homology (CH) domain from residues 1–130. This region mainly exists as monomer 14 , 25 and is crucial for microtubule binding 8 , 26 . Specifically, the positively charged amino acids of this domain are critical for microtubule binding 8 , 27 , 28 .…”

Section: Introductionmentioning

confidence: 99%

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

Gireesh

Shine

Lakshmi

et al. 2018

Sci Rep

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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.

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Section: Introductionsupporting

confidence: 56%

Section: Resultsmentioning

confidence: 99%

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

Gireesh

Shine

Lakshmi

et al. 2018

Sci Rep

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“…Recent work with GTPγS microtubules shows that they are not stiff like GMPCPP microtubules . Further, end‐binding proteins (EB) that tip‐track growing microtubules have been shown to have enhanced binding for GTPγS microtubules . Taken together, these results point to an altered conformational state for GTPγS compared to GTP, GMPCPP, or GDP.…”

Section: Gtpase Filaments: Microtubules and Tubulin Structure And Funmentioning

confidence: 99%

Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading

Bailey¹,

Jiang

Dima

et al. 2016

Biopolymers

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Microtubules are amazing filaments made of GTPase enzymes that store energy used for their own self-destruction to cause a stochastically driven dynamics called dynamic instability. Dynamic instability can be reproduced in vitro with purified tubulin, but the dynamics do not mimic that observed in cells. This is because stabilizers and destabilizers act to alter microtubule dynamics. One interesting and understudied class of destabilizers consists of the microtubule-severing enzymes from the ATPases Associated with various cellular Activities (AAA+) family of ATP-enzymes. Here we review current knowledge about GTP-driven microtubule dynamics and how that couples to ATP-driven destabilization by severing enzymes. We present a list of challenges regarding the mechanism of severing, which require development of experimental and modeling approaches to shed light as to how severing enzymes can act to regulate microtubule dynamics in cells. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 547-556, 2016.

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“…In cells, CYLD acts in concert with the +TIPs protein EB1 to regulate microtubule dynamics [ 22 ]. Recent study reveals that EB1 can form dimers by an intermolecular interaction, which leads to the autoinhibition of EB1 activity [ 19 , 23 ]. The dimerization-caused autoinhibition of EB1 prompts us to investigate whether CYLD has similar behavior.…”

Section: Resultsmentioning

confidence: 99%

The B-box module of CYLD is responsible for its intermolecular interaction and cytoplasmic localization

et al. 2017

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The tumor suppressor protein cylindromatosis (CYLD), as a microtubule-associated deubiquitinase, plays a pivotal role in a wide range of cellular activities, including innate immunity, cell division, and ciliogenesis. Structural characterization reveals a small zinc-binding B-box inserted within the ubiquitin specific protease (USP) domain of CYLD; however, the exact role for this module remains yet to be elucidated. Here we identify a critical role for the B-box in facilitating the intermolecular interaction and subcellular localization of CYLD. By co-immunoprecipitation assays we uncover that CYLD has the ability to form an intermolecular complex. Native gel electrophoresis analysis and pull down assays show that the USP domain of CYLD is essential for its intermolecular interaction. Further investigation reveals that deletion of the B-box from the USP domain disrupts the intermolecular interaction of CYLD. Importantly, although loss of the B-box has no obvious effect on the deubiquitinase activity of CYLD, it abolishes the USP domain-mediated retention of CYLD in the cytoplasm. Collectively, these data demonstrate an important role for the B-box module of CYLD in mediating its assembly and subcellular distribution, which might be related to the functions of CYLD in various biological processes.

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Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR

Cited by 11 publications

References 27 publications

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

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

Invited review: Microtubule severing enzymes couple atpase activity with tubulin GTPase spring loading

The B-box module of CYLD is responsible for its intermolecular interaction and cytoplasmic localization

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