Limits...
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.

Show MeSH

Related in: MedlinePlus

Mal3 Binds between Microtubule Protofilaments except at the Seam(A) 15 Å cryo-EM reconstruction of 13-protofilament Mal3143-GTPγS microtubules, displayed with the microtubule plus end oriented up. Mal3143 (green) binds the cleft between protofilaments (gray) making B lattice contacts.(B) Same reconstruction as in (A) but rotated 180° around the microtubule axis. The A lattice seam is only marginally occupied.See also Figure S1.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3368265&req=5

fig1: Mal3 Binds between Microtubule Protofilaments except at the Seam(A) 15 Å cryo-EM reconstruction of 13-protofilament Mal3143-GTPγS microtubules, displayed with the microtubule plus end oriented up. Mal3143 (green) binds the cleft between protofilaments (gray) making B lattice contacts.(B) Same reconstruction as in (A) but rotated 180° around the microtubule axis. The A lattice seam is only marginally occupied.See also Figure S1.

Mentions: We used cryo-EM to determine how the CH domain of Mal3, the fission yeast EB, binds to GTPγS microtubules, which were previously shown to be static mimics of growing microtubule ends (Maurer et al., 2011). We found that Mal3 favors the assembly of GTPγS-tubulin into microtubules with mostly 13 protofilaments (Figure S1B available online), consistent with a previous study of EB1 in presence of GTP-tubulin (Vitre et al., 2008). Three-dimensional (3D) reconstruction from segments of 13 protofilament GTPγS microtubules decorated with an N-terminal fragment of Mal3 containing its CH domain (Mal3143) generated an asymmetric structure with 15 Å resolution (Figures 1, S1C, and S1D). Mal3143 binds regularly between neighboring protofilaments (B lattice, 12 such contacts per 13 protofilament microtubule) except along the seam (the single A lattice contact between protofilaments) of GTPγS microtubules (Figure 1B). This selectivity suggests a highly specific binding site. The longitudinal distance between bound CH domains is 8 nm (corresponding to one tubulin heterodimer), resulting in a stoichiometry of 12 CH domains per 13 tubulin dimers. This is in good agreement with the recently reported approximately stoichiometric binding measured by fluorescence microscopy (Maurer et al., 2011). A similar pattern of binding—including absence of interaction at the seam—was seen for Mal3143 on GDP microtubules (Figures S1E–S1H). This is in contrast to previous reports (des Georges et al., 2008; Sandblad et al., 2006) (see Discussion). Our data suggest that the affinity difference of Mal3 binding to growing microtubule ends compared to older parts of the microtubule lattice (Maurer et al., 2011) is not a result of different binding positions nor of dramatic A-to-B lattice transitions within the microtubule but rather a consequence of conformational rearrangements within its binding site.


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

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

Mal3 Binds between Microtubule Protofilaments except at the Seam(A) 15 Å cryo-EM reconstruction of 13-protofilament Mal3143-GTPγS microtubules, displayed with the microtubule plus end oriented up. Mal3143 (green) binds the cleft between protofilaments (gray) making B lattice contacts.(B) Same reconstruction as in (A) but rotated 180° around the microtubule axis. The A lattice seam is only marginally occupied.See also Figure S1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368265&req=5

fig1: Mal3 Binds between Microtubule Protofilaments except at the Seam(A) 15 Å cryo-EM reconstruction of 13-protofilament Mal3143-GTPγS microtubules, displayed with the microtubule plus end oriented up. Mal3143 (green) binds the cleft between protofilaments (gray) making B lattice contacts.(B) Same reconstruction as in (A) but rotated 180° around the microtubule axis. The A lattice seam is only marginally occupied.See also Figure S1.
Mentions: We used cryo-EM to determine how the CH domain of Mal3, the fission yeast EB, binds to GTPγS microtubules, which were previously shown to be static mimics of growing microtubule ends (Maurer et al., 2011). We found that Mal3 favors the assembly of GTPγS-tubulin into microtubules with mostly 13 protofilaments (Figure S1B available online), consistent with a previous study of EB1 in presence of GTP-tubulin (Vitre et al., 2008). Three-dimensional (3D) reconstruction from segments of 13 protofilament GTPγS microtubules decorated with an N-terminal fragment of Mal3 containing its CH domain (Mal3143) generated an asymmetric structure with 15 Å resolution (Figures 1, S1C, and S1D). Mal3143 binds regularly between neighboring protofilaments (B lattice, 12 such contacts per 13 protofilament microtubule) except along the seam (the single A lattice contact between protofilaments) of GTPγS microtubules (Figure 1B). This selectivity suggests a highly specific binding site. The longitudinal distance between bound CH domains is 8 nm (corresponding to one tubulin heterodimer), resulting in a stoichiometry of 12 CH domains per 13 tubulin dimers. This is in good agreement with the recently reported approximately stoichiometric binding measured by fluorescence microscopy (Maurer et al., 2011). A similar pattern of binding—including absence of interaction at the seam—was seen for Mal3143 on GDP microtubules (Figures S1E–S1H). This is in contrast to previous reports (des Georges et al., 2008; Sandblad et al., 2006) (see Discussion). Our data suggest that the affinity difference of Mal3 binding to growing microtubule ends compared to older parts of the microtubule lattice (Maurer et al., 2011) is not a result of different binding positions nor of dramatic A-to-B lattice transitions within the microtubule but rather a consequence of conformational rearrangements within its binding site.

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