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EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.

Maurer SP, Fourniol FJ, Bohner G, Moores CA, Surrey T - Cell (2012)

Bottom Line: By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state.The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization.This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.

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Comparison of Lateral Tubulin-Tubulin B Lattice Contacts in GTPγS and GDP Lattices, Related to Figure 6Comparison of the cryo-EM maps of Mal3-decorated GTPγS microtubules (top, gray mesh, 8.6 Å resolution, see also Figure 6) and GDP microtubules stabilized by doublecortin (middle, blue surface; EMDB 1788; Fourniol et al., 2010; 8.2 Å resolution). Longitudinal sections of the EM map illustrating the two layers of tubulin-tubulin lateral contacts: additional H3-H9 contacts are observed only in GTPγS microtubules (left), whereas the conserved contacts involving the M loop, N loop, and H2-S3 loops of tubulin (Sui and Downing, 2010) are observed in both GTPγS and GDP microtubules (right). Interestingly, although the additional H3-H9 contacts in GTPγS microtubules appear to be more pronounced for neighboring β-tubulin contacts, they are also visible between neighboring α-tubulins, suggesting that concerted conformational changes take place in the GTPγS lattice-incorporated tubulin heterodimers. The overlays (bottom) illustrate the good agreement between the two density maps displayed with an equivalent contouring level representative of the whole protein complex volume (GTPγS map rendered at a threshold of 1.68 σ; DCX-GDP map rendered at a threshold of 0.78 σ), and highlights the differences in density due to H3-H9 contacts (circled, the β-β contact extends over ∼10 Å, the α-α contact over ∼5 Å).
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figs5: Comparison of Lateral Tubulin-Tubulin B Lattice Contacts in GTPγS and GDP Lattices, Related to Figure 6Comparison of the cryo-EM maps of Mal3-decorated GTPγS microtubules (top, gray mesh, 8.6 Å resolution, see also Figure 6) and GDP microtubules stabilized by doublecortin (middle, blue surface; EMDB 1788; Fourniol et al., 2010; 8.2 Å resolution). Longitudinal sections of the EM map illustrating the two layers of tubulin-tubulin lateral contacts: additional H3-H9 contacts are observed only in GTPγS microtubules (left), whereas the conserved contacts involving the M loop, N loop, and H2-S3 loops of tubulin (Sui and Downing, 2010) are observed in both GTPγS and GDP microtubules (right). Interestingly, although the additional H3-H9 contacts in GTPγS microtubules appear to be more pronounced for neighboring β-tubulin contacts, they are also visible between neighboring α-tubulins, suggesting that concerted conformational changes take place in the GTPγS lattice-incorporated tubulin heterodimers. The overlays (bottom) illustrate the good agreement between the two density maps displayed with an equivalent contouring level representative of the whole protein complex volume (GTPγS map rendered at a threshold of 1.68 σ; DCX-GDP map rendered at a threshold of 0.78 σ), and highlights the differences in density due to H3-H9 contacts (circled, the β-β contact extends over ∼10 Å, the α-α contact over ∼5 Å).

Mentions: In Mal3143-decorated GTPγS microtubules, as in all subnanometer microtubule reconstructions reported to date (Fourniol et al., 2010; Li et al., 2002; Sui and Downing, 2010), interprotofilament lateral contacts that involve secondary structure elements facing the microtubule lumen are observed: the M loop (S7-H9) of one subunit contacts the N loop (H1-S2) and H2-S3 loop of the neighboring tubulin subunit (Figure S5). However, strikingly, we also observed an enhanced layer of lateral contacts at higher radius in GTPγS microtubules (Figures 6B and S5). These enhanced lateral contacts involve tubulin helices H3 that adjoin H9 of neighboring tubulins, presumably because of a structural change—possibly a positional shift—in the H3 helix of β-tubulin in the GTPγS microtubule. This might be part of the structural alteration that is sensed by EBs. A similar but smaller lateral contact between α-tubulins (Figure S5) is likely to result from cooperative conformational rearrangements within the lattice. The importance of the H3 contacts in our structure is consistent with alanine scanning mutagenesis in yeast, showing that mutations in α- and β-tubulin H3 cause temperature sensitivity (Reijo et al., 1994; Richards et al., 2000). The structural change in the β-tubulin H3 helix could be triggered by nucleotide hydrolysis, sensed by H3 via loop T3, which might be mimicked by the presence of a bulky group such as the γ-S-phosphate (or BeF3−; Maurer et al., 2011) occupying the γ-phosphate-binding pocket. EBs appear therefore to be optimally positioned to sense nucleotide hydrolysis-dependent structural changes in the microtubule lattice.


EBs recognize a nucleotide-dependent structural cap at growing microtubule ends.

Maurer SP, Fourniol FJ, Bohner G, Moores CA, Surrey T - Cell (2012)

Comparison of Lateral Tubulin-Tubulin B Lattice Contacts in GTPγS and GDP Lattices, Related to Figure 6Comparison of the cryo-EM maps of Mal3-decorated GTPγS microtubules (top, gray mesh, 8.6 Å resolution, see also Figure 6) and GDP microtubules stabilized by doublecortin (middle, blue surface; EMDB 1788; Fourniol et al., 2010; 8.2 Å resolution). Longitudinal sections of the EM map illustrating the two layers of tubulin-tubulin lateral contacts: additional H3-H9 contacts are observed only in GTPγS microtubules (left), whereas the conserved contacts involving the M loop, N loop, and H2-S3 loops of tubulin (Sui and Downing, 2010) are observed in both GTPγS and GDP microtubules (right). Interestingly, although the additional H3-H9 contacts in GTPγS microtubules appear to be more pronounced for neighboring β-tubulin contacts, they are also visible between neighboring α-tubulins, suggesting that concerted conformational changes take place in the GTPγS lattice-incorporated tubulin heterodimers. The overlays (bottom) illustrate the good agreement between the two density maps displayed with an equivalent contouring level representative of the whole protein complex volume (GTPγS map rendered at a threshold of 1.68 σ; DCX-GDP map rendered at a threshold of 0.78 σ), and highlights the differences in density due to H3-H9 contacts (circled, the β-β contact extends over ∼10 Å, the α-α contact over ∼5 Å).
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Related In: Results  -  Collection

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figs5: Comparison of Lateral Tubulin-Tubulin B Lattice Contacts in GTPγS and GDP Lattices, Related to Figure 6Comparison of the cryo-EM maps of Mal3-decorated GTPγS microtubules (top, gray mesh, 8.6 Å resolution, see also Figure 6) and GDP microtubules stabilized by doublecortin (middle, blue surface; EMDB 1788; Fourniol et al., 2010; 8.2 Å resolution). Longitudinal sections of the EM map illustrating the two layers of tubulin-tubulin lateral contacts: additional H3-H9 contacts are observed only in GTPγS microtubules (left), whereas the conserved contacts involving the M loop, N loop, and H2-S3 loops of tubulin (Sui and Downing, 2010) are observed in both GTPγS and GDP microtubules (right). Interestingly, although the additional H3-H9 contacts in GTPγS microtubules appear to be more pronounced for neighboring β-tubulin contacts, they are also visible between neighboring α-tubulins, suggesting that concerted conformational changes take place in the GTPγS lattice-incorporated tubulin heterodimers. The overlays (bottom) illustrate the good agreement between the two density maps displayed with an equivalent contouring level representative of the whole protein complex volume (GTPγS map rendered at a threshold of 1.68 σ; DCX-GDP map rendered at a threshold of 0.78 σ), and highlights the differences in density due to H3-H9 contacts (circled, the β-β contact extends over ∼10 Å, the α-α contact over ∼5 Å).
Mentions: In Mal3143-decorated GTPγS microtubules, as in all subnanometer microtubule reconstructions reported to date (Fourniol et al., 2010; Li et al., 2002; Sui and Downing, 2010), interprotofilament lateral contacts that involve secondary structure elements facing the microtubule lumen are observed: the M loop (S7-H9) of one subunit contacts the N loop (H1-S2) and H2-S3 loop of the neighboring tubulin subunit (Figure S5). However, strikingly, we also observed an enhanced layer of lateral contacts at higher radius in GTPγS microtubules (Figures 6B and S5). These enhanced lateral contacts involve tubulin helices H3 that adjoin H9 of neighboring tubulins, presumably because of a structural change—possibly a positional shift—in the H3 helix of β-tubulin in the GTPγS microtubule. This might be part of the structural alteration that is sensed by EBs. A similar but smaller lateral contact between α-tubulins (Figure S5) is likely to result from cooperative conformational rearrangements within the lattice. The importance of the H3 contacts in our structure is consistent with alanine scanning mutagenesis in yeast, showing that mutations in α- and β-tubulin H3 cause temperature sensitivity (Reijo et al., 1994; Richards et al., 2000). The structural change in the β-tubulin H3 helix could be triggered by nucleotide hydrolysis, sensed by H3 via loop T3, which might be mimicked by the presence of a bulky group such as the γ-S-phosphate (or BeF3−; Maurer et al., 2011) occupying the γ-phosphate-binding pocket. EBs appear therefore to be optimally positioned to sense nucleotide hydrolysis-dependent structural changes in the microtubule lattice.

Bottom Line: By binding close to the exchangeable GTP-binding site, the CH domain is ideally positioned to sense the microtubule's nucleotide state.The same microtubule-end region is also a stabilizing structural cap protecting the microtubule from depolymerization.This insight supports a common structural link between two important biological phenomena, microtubule dynamic instability and end tracking.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3LY, UK.

Show MeSH
Related in: MedlinePlus