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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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The Dam1 complex MT-binding domain.Structural models show that the C-terminal regions of Dam1p and Duo1p interact with the N-terminal regions of Dam1p and Duo1p in the absence of MTs. On binding MTs, these interactions are lost, freeing the C-terminal regions of Dam1 and Duo1, and allowing them to bind the MT. (a) Dam1N (light yellow) to Dam1C (dark yellow); (b) Duo1N (turquoise) to Duo1C (blue); (c) Dam1C (dark yellow) to Duo1N (turquoise); (d) Dam1N (light yellow) to Duo1C (blue). The Dam1 complex is shown as a monomer.
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f6: The Dam1 complex MT-binding domain.Structural models show that the C-terminal regions of Dam1p and Duo1p interact with the N-terminal regions of Dam1p and Duo1p in the absence of MTs. On binding MTs, these interactions are lost, freeing the C-terminal regions of Dam1 and Duo1, and allowing them to bind the MT. (a) Dam1N (light yellow) to Dam1C (dark yellow); (b) Duo1N (turquoise) to Duo1C (blue); (c) Dam1C (dark yellow) to Duo1N (turquoise); (d) Dam1N (light yellow) to Duo1C (blue). The Dam1 complex is shown as a monomer.

Mentions: To examine these conformational changes in more detail, we analysed the interactions at a coarse structural level. We divided each protein into ‘macro-regions' of ∼100 amino acid residues (Supplementary Table 12). For example, Ask1p was divided into three macro-regions: Ask1N is the N-terminal 97 amino acids, Ask1M is the middle 97 amino acids and Ask1C is the C-terminal 98 amino acids. Proteins under 100 amino acids, such as Dad1p, Dad2p and so on consisted of just one macro-region: the whole protein. We then compared the interactions of each macro-region in the presence and absence of MTs (Supplementary Table 13). A striking difference is seen for Dam1C and Duo1C. In the absence of MTs, Dam1C and Duo1C interact with Dam1N and Duo1N. When bound to MTs, these interactions are not present (Fig. 6 and Supplementary Table 13, grey highlighting). Instead, Dam1C and Duo1C move to the surface of the complex and bind to the MT (Supplementary Movie 4).


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)

The Dam1 complex MT-binding domain.Structural models show that the C-terminal regions of Dam1p and Duo1p interact with the N-terminal regions of Dam1p and Duo1p in the absence of MTs. On binding MTs, these interactions are lost, freeing the C-terminal regions of Dam1 and Duo1, and allowing them to bind the MT. (a) Dam1N (light yellow) to Dam1C (dark yellow); (b) Duo1N (turquoise) to Duo1C (blue); (c) Dam1C (dark yellow) to Duo1N (turquoise); (d) Dam1N (light yellow) to Duo1C (blue). The Dam1 complex is shown as a monomer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: The Dam1 complex MT-binding domain.Structural models show that the C-terminal regions of Dam1p and Duo1p interact with the N-terminal regions of Dam1p and Duo1p in the absence of MTs. On binding MTs, these interactions are lost, freeing the C-terminal regions of Dam1 and Duo1, and allowing them to bind the MT. (a) Dam1N (light yellow) to Dam1C (dark yellow); (b) Duo1N (turquoise) to Duo1C (blue); (c) Dam1C (dark yellow) to Duo1N (turquoise); (d) Dam1N (light yellow) to Duo1C (blue). The Dam1 complex is shown as a monomer.
Mentions: To examine these conformational changes in more detail, we analysed the interactions at a coarse structural level. We divided each protein into ‘macro-regions' of ∼100 amino acid residues (Supplementary Table 12). For example, Ask1p was divided into three macro-regions: Ask1N is the N-terminal 97 amino acids, Ask1M is the middle 97 amino acids and Ask1C is the C-terminal 98 amino acids. Proteins under 100 amino acids, such as Dad1p, Dad2p and so on consisted of just one macro-region: the whole protein. We then compared the interactions of each macro-region in the presence and absence of MTs (Supplementary Table 13). A striking difference is seen for Dam1C and Duo1C. In the absence of MTs, Dam1C and Duo1C interact with Dam1N and Duo1N. When bound to MTs, these interactions are not present (Fig. 6 and Supplementary Table 13, grey highlighting). Instead, Dam1C and Duo1C move to the surface of the complex and bind to the MT (Supplementary Movie 4).

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
Related in: MedlinePlus