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The molecular architecture of the Dam1 kinetochore complex is defined by cross-linking based structural modelling.

Zelter A, Bonomi M, Kim Jo, Umbreit NT, Hoopmann MR, Johnson R, Riffle M, Jaschob D, MacCoss MJ, Moritz RL, Davis TN - Nat Commun (2015)

Bottom Line: Aurora B phosphorylation of Dam1p C terminus weakens direct interaction with the microtubule.Furthermore, the Dam1p amino terminus forms an interaction interface between Dam1 complexes, which is also disrupted by phosphorylation.Our results demonstrate that Aurora B inhibits both direct interaction with the microtubule and oligomerization of the Dam1 complex to drive error correction during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Accurate segregation of chromosomes during cell division is essential. The Dam1 complex binds kinetochores to microtubules and its oligomerization is required to form strong attachments. It is a key target of Aurora B kinase, which destabilizes erroneous attachments allowing subsequent correction. Understanding the roles and regulation of the Dam1 complex requires structural information. Here we apply cross-linking/mass spectrometry and structural modelling to determine the molecular architecture of the Dam1 complex. We find microtubule attachment is accompanied by substantial conformational changes, with direct binding mediated by the carboxy termini of Dam1p and Duo1p. Aurora B phosphorylation of Dam1p C terminus weakens direct interaction with the microtubule. Furthermore, the Dam1p amino terminus forms an interaction interface between Dam1 complexes, which is also disrupted by phosphorylation. Our results demonstrate that Aurora B inhibits both direct interaction with the microtubule and oligomerization of the Dam1 complex to drive error correction during mitosis.

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Cross-link based flexible docking of the Dam1 complex model onto the MT specifies a preferred orientation.(a) The model of the Dam1 complex on the MT with beads coloured by protein (Hsk1p is not visible at this angle). (b) Only those beads found cross-linked to the MT are highlighted. (c) The orientation of the Dam1 complex on the MT with respect to the orientation of the Ndc80 complex. The C-terminal MT-binding region of the Ndc80 complex is nearer the minus end of the MT. Aravamudhan et al.29 measured FRET between N-Nuf2 and six Dam1 complex C termini29. The proteins in the key to the left and right of the model represent these results, which indicates proximity between the C termini of Dam1p, Dad4p and Dad3p to the N terminus of Nuf2, while suggesting that the C termini of Spc34p, Ask1p and Dad1p are further away. Only a Dam1 complex monomer is shown for clarity. (d) Attempts to dock the model of the Dam1 complex in the minus MT conformation resulted in a poor fit and an orientation incompatible with a ring.
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f5: Cross-link based flexible docking of the Dam1 complex model onto the MT specifies a preferred orientation.(a) The model of the Dam1 complex on the MT with beads coloured by protein (Hsk1p is not visible at this angle). (b) Only those beads found cross-linked to the MT are highlighted. (c) The orientation of the Dam1 complex on the MT with respect to the orientation of the Ndc80 complex. The C-terminal MT-binding region of the Ndc80 complex is nearer the minus end of the MT. Aravamudhan et al.29 measured FRET between N-Nuf2 and six Dam1 complex C termini29. The proteins in the key to the left and right of the model represent these results, which indicates proximity between the C termini of Dam1p, Dad4p and Dad3p to the N terminus of Nuf2, while suggesting that the C termini of Spc34p, Ask1p and Dad1p are further away. Only a Dam1 complex monomer is shown for clarity. (d) Attempts to dock the model of the Dam1 complex in the minus MT conformation resulted in a poor fit and an orientation incompatible with a ring.

Mentions: We then used a flexible-docking procedure to fit this model of the Dam1 complex dimer onto a MT, based on the cross-links observed between the Dam1 complex and tubulin (Figs 4,5, Supplementary Movie 3 and Supplementary File 3). A good fit was obtained, as 76% of the observed Dam1 complex to tubulin cross-links were satisfied (Supplementary Table 11). Furthermore, the top 3,264 models produced a single preferred orientation with respect to MT polarity (as depicted in Fig. 5), while alternative orientations were only found in low-scoring models. The orientation observed is compatible with ring formation, as the Dam1 dimer is arranged perpendicular to the protofilaments. Moreover, the orientation is consistent with recently published in vivo fluorescence resonance energy transfer (FRET) data29 (Fig. 5c).


The molecular architecture of the Dam1 kinetochore complex is defined by cross-linking based structural modelling.

Zelter A, Bonomi M, Kim Jo, Umbreit NT, Hoopmann MR, Johnson R, Riffle M, Jaschob D, MacCoss MJ, Moritz RL, Davis TN - Nat Commun (2015)

Cross-link based flexible docking of the Dam1 complex model onto the MT specifies a preferred orientation.(a) The model of the Dam1 complex on the MT with beads coloured by protein (Hsk1p is not visible at this angle). (b) Only those beads found cross-linked to the MT are highlighted. (c) The orientation of the Dam1 complex on the MT with respect to the orientation of the Ndc80 complex. The C-terminal MT-binding region of the Ndc80 complex is nearer the minus end of the MT. Aravamudhan et al.29 measured FRET between N-Nuf2 and six Dam1 complex C termini29. The proteins in the key to the left and right of the model represent these results, which indicates proximity between the C termini of Dam1p, Dad4p and Dad3p to the N terminus of Nuf2, while suggesting that the C termini of Spc34p, Ask1p and Dad1p are further away. Only a Dam1 complex monomer is shown for clarity. (d) Attempts to dock the model of the Dam1 complex in the minus MT conformation resulted in a poor fit and an orientation incompatible with a ring.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660060&req=5

f5: Cross-link based flexible docking of the Dam1 complex model onto the MT specifies a preferred orientation.(a) The model of the Dam1 complex on the MT with beads coloured by protein (Hsk1p is not visible at this angle). (b) Only those beads found cross-linked to the MT are highlighted. (c) The orientation of the Dam1 complex on the MT with respect to the orientation of the Ndc80 complex. The C-terminal MT-binding region of the Ndc80 complex is nearer the minus end of the MT. Aravamudhan et al.29 measured FRET between N-Nuf2 and six Dam1 complex C termini29. The proteins in the key to the left and right of the model represent these results, which indicates proximity between the C termini of Dam1p, Dad4p and Dad3p to the N terminus of Nuf2, while suggesting that the C termini of Spc34p, Ask1p and Dad1p are further away. Only a Dam1 complex monomer is shown for clarity. (d) Attempts to dock the model of the Dam1 complex in the minus MT conformation resulted in a poor fit and an orientation incompatible with a ring.
Mentions: We then used a flexible-docking procedure to fit this model of the Dam1 complex dimer onto a MT, based on the cross-links observed between the Dam1 complex and tubulin (Figs 4,5, Supplementary Movie 3 and Supplementary File 3). A good fit was obtained, as 76% of the observed Dam1 complex to tubulin cross-links were satisfied (Supplementary Table 11). Furthermore, the top 3,264 models produced a single preferred orientation with respect to MT polarity (as depicted in Fig. 5), while alternative orientations were only found in low-scoring models. The orientation observed is compatible with ring formation, as the Dam1 dimer is arranged perpendicular to the protofilaments. Moreover, the orientation is consistent with recently published in vivo fluorescence resonance energy transfer (FRET) data29 (Fig. 5c).

Bottom Line: Aurora B phosphorylation of Dam1p C terminus weakens direct interaction with the microtubule.Furthermore, the Dam1p amino terminus forms an interaction interface between Dam1 complexes, which is also disrupted by phosphorylation.Our results demonstrate that Aurora B inhibits both direct interaction with the microtubule and oligomerization of the Dam1 complex to drive error correction during mitosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

ABSTRACT
Accurate segregation of chromosomes during cell division is essential. The Dam1 complex binds kinetochores to microtubules and its oligomerization is required to form strong attachments. It is a key target of Aurora B kinase, which destabilizes erroneous attachments allowing subsequent correction. Understanding the roles and regulation of the Dam1 complex requires structural information. Here we apply cross-linking/mass spectrometry and structural modelling to determine the molecular architecture of the Dam1 complex. We find microtubule attachment is accompanied by substantial conformational changes, with direct binding mediated by the carboxy termini of Dam1p and Duo1p. Aurora B phosphorylation of Dam1p C terminus weakens direct interaction with the microtubule. Furthermore, the Dam1p amino terminus forms an interaction interface between Dam1 complexes, which is also disrupted by phosphorylation. Our results demonstrate that Aurora B inhibits both direct interaction with the microtubule and oligomerization of the Dam1 complex to drive error correction during mitosis.

Show MeSH