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Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer.

Yang M, Li B, Liu CJ, Tomchick DR, Machius M, Rizo J, Yu H, Luo X - PLoS Biol. (2008)

Bottom Line: A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells.Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro.Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C(Cdc20) inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2-C-Mad2 (C-C) or asymmetric O-Mad2-C-Mad2 (O-C) dimers. We also report the crystal structure of the symmetric C-C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells. Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

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Interactions at the Symmetric Mad2 Dimer Interface(A and B) Interactions between Mad2A and Mad2B. The side chains of contacting residues are shown as sticks. Nitrogen and oxygen atoms are colored blue and red, respectively. Mad2A carbons are colored yellow; Mad2B carbons are colored gray and labeled in italics. The tightly bound water molecules are drawn as red spheres.(C) Surface diagram of the Mad2L13A dimer. Same color scheme is used as in Figure 3A–3C.(D) Hydrogen bonds with Q134 in Mad2A. Hydrogen bonds are indicated by red dashed lines.(E) Mad2R184E adopts the monomeric C-Mad2 conformation. Gel filtration chromatogram of the high-salt peak (275 mM salt, Q2) of Mad2R184E obtained with anion exchange chromatography is shown. Mad2R184E has an apparent molecular mass of about 30 kDa, which is consistent with it being a monomer. The elution profile for Mad2R184E is shown in red, and the elution profile for molecular weight standards is shown in gray. The positions for 44 kDa and 17 kDa markers are indicated.(F) The high-field methyl region of the 1D 1H spectrum of Mad2R184E. The V197γ2 peak (−0.34 ppm) specific to C-Mad2 is labeled. The line width of the methyl peaks is consistent with C-Mad2R184E being monomeric.
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pbio-0060050-g004: Interactions at the Symmetric Mad2 Dimer Interface(A and B) Interactions between Mad2A and Mad2B. The side chains of contacting residues are shown as sticks. Nitrogen and oxygen atoms are colored blue and red, respectively. Mad2A carbons are colored yellow; Mad2B carbons are colored gray and labeled in italics. The tightly bound water molecules are drawn as red spheres.(C) Surface diagram of the Mad2L13A dimer. Same color scheme is used as in Figure 3A–3C.(D) Hydrogen bonds with Q134 in Mad2A. Hydrogen bonds are indicated by red dashed lines.(E) Mad2R184E adopts the monomeric C-Mad2 conformation. Gel filtration chromatogram of the high-salt peak (275 mM salt, Q2) of Mad2R184E obtained with anion exchange chromatography is shown. Mad2R184E has an apparent molecular mass of about 30 kDa, which is consistent with it being a monomer. The elution profile for Mad2R184E is shown in red, and the elution profile for molecular weight standards is shown in gray. The positions for 44 kDa and 17 kDa markers are indicated.(F) The high-field methyl region of the 1D 1H spectrum of Mad2R184E. The V197γ2 peak (−0.34 ppm) specific to C-Mad2 is labeled. The line width of the methyl peaks is consistent with C-Mad2R184E being monomeric.

Mentions: Both monomers in the Mad2L13A dimer adopt the C-Mad2 conformation and are related by noncrystallographic, two-fold symmetry (Figures 3A–3C and S3). The two monomers mainly interact through the C-terminal halves of their αC helices. The high resolution of our structure of Mad2L13A allows clear visualization of side chains as well as several well-ordered water molecules at the dimer interface (Figure 3D). The dimerization interface of Mad2L13A is symmetric and consists of residues from the C-terminal half of αC, R184 from β8′, and Q34 at the C-terminal end of αA (Figure 4). These residues form hydrophobic interactions and extensive networks of water-mediated hydrogen bonds. For example, F141 forms intermolecular interactions with A137, T138, Q134, and F141 (Figure 4A). Bridged by two tightly bound water molecules, R133 from one monomer forms a network of hydrogen bonds with both the backbone and side-chain carbonyl groups of Q34 and the backbone carbonyl of T136 from the neighboring monomer (Figure 4B). The interactions between the two Mad2 monomers observed in our structure are consistent with previous mutagenesis results [23]. Mutations of residues directly located at the dimer interface, including R133, Q134, T140, and F141, have been shown to disrupt Mad2 dimerization.


Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer.

Yang M, Li B, Liu CJ, Tomchick DR, Machius M, Rizo J, Yu H, Luo X - PLoS Biol. (2008)

Interactions at the Symmetric Mad2 Dimer Interface(A and B) Interactions between Mad2A and Mad2B. The side chains of contacting residues are shown as sticks. Nitrogen and oxygen atoms are colored blue and red, respectively. Mad2A carbons are colored yellow; Mad2B carbons are colored gray and labeled in italics. The tightly bound water molecules are drawn as red spheres.(C) Surface diagram of the Mad2L13A dimer. Same color scheme is used as in Figure 3A–3C.(D) Hydrogen bonds with Q134 in Mad2A. Hydrogen bonds are indicated by red dashed lines.(E) Mad2R184E adopts the monomeric C-Mad2 conformation. Gel filtration chromatogram of the high-salt peak (275 mM salt, Q2) of Mad2R184E obtained with anion exchange chromatography is shown. Mad2R184E has an apparent molecular mass of about 30 kDa, which is consistent with it being a monomer. The elution profile for Mad2R184E is shown in red, and the elution profile for molecular weight standards is shown in gray. The positions for 44 kDa and 17 kDa markers are indicated.(F) The high-field methyl region of the 1D 1H spectrum of Mad2R184E. The V197γ2 peak (−0.34 ppm) specific to C-Mad2 is labeled. The line width of the methyl peaks is consistent with C-Mad2R184E being monomeric.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2270309&req=5

pbio-0060050-g004: Interactions at the Symmetric Mad2 Dimer Interface(A and B) Interactions between Mad2A and Mad2B. The side chains of contacting residues are shown as sticks. Nitrogen and oxygen atoms are colored blue and red, respectively. Mad2A carbons are colored yellow; Mad2B carbons are colored gray and labeled in italics. The tightly bound water molecules are drawn as red spheres.(C) Surface diagram of the Mad2L13A dimer. Same color scheme is used as in Figure 3A–3C.(D) Hydrogen bonds with Q134 in Mad2A. Hydrogen bonds are indicated by red dashed lines.(E) Mad2R184E adopts the monomeric C-Mad2 conformation. Gel filtration chromatogram of the high-salt peak (275 mM salt, Q2) of Mad2R184E obtained with anion exchange chromatography is shown. Mad2R184E has an apparent molecular mass of about 30 kDa, which is consistent with it being a monomer. The elution profile for Mad2R184E is shown in red, and the elution profile for molecular weight standards is shown in gray. The positions for 44 kDa and 17 kDa markers are indicated.(F) The high-field methyl region of the 1D 1H spectrum of Mad2R184E. The V197γ2 peak (−0.34 ppm) specific to C-Mad2 is labeled. The line width of the methyl peaks is consistent with C-Mad2R184E being monomeric.
Mentions: Both monomers in the Mad2L13A dimer adopt the C-Mad2 conformation and are related by noncrystallographic, two-fold symmetry (Figures 3A–3C and S3). The two monomers mainly interact through the C-terminal halves of their αC helices. The high resolution of our structure of Mad2L13A allows clear visualization of side chains as well as several well-ordered water molecules at the dimer interface (Figure 3D). The dimerization interface of Mad2L13A is symmetric and consists of residues from the C-terminal half of αC, R184 from β8′, and Q34 at the C-terminal end of αA (Figure 4). These residues form hydrophobic interactions and extensive networks of water-mediated hydrogen bonds. For example, F141 forms intermolecular interactions with A137, T138, Q134, and F141 (Figure 4A). Bridged by two tightly bound water molecules, R133 from one monomer forms a network of hydrogen bonds with both the backbone and side-chain carbonyl groups of Q34 and the backbone carbonyl of T136 from the neighboring monomer (Figure 4B). The interactions between the two Mad2 monomers observed in our structure are consistent with previous mutagenesis results [23]. Mutations of residues directly located at the dimer interface, including R133, Q134, T140, and F141, have been shown to disrupt Mad2 dimerization.

Bottom Line: A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells.Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro.Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States of America.

ABSTRACT
In response to misaligned sister chromatids during mitosis, the spindle checkpoint protein Mad2 inhibits the anaphase-promoting complex or cyclosome (APC/C) through binding to its mitotic activator Cdc20, thus delaying anaphase onset. Mad1, an upstream regulator of Mad2, forms a tight core complex with Mad2 and facilitates Mad2 binding to Cdc20. In the absence of its binding proteins, free Mad2 has two natively folded conformers, termed N1-Mad2/open-Mad2 (O-Mad2) and N2-Mad2/closed Mad2 (C-Mad2), with C-Mad2 being more active in APC/C(Cdc20) inhibition. Here, we show that whereas O-Mad2 is monomeric, C-Mad2 forms either symmetric C-Mad2-C-Mad2 (C-C) or asymmetric O-Mad2-C-Mad2 (O-C) dimers. We also report the crystal structure of the symmetric C-C Mad2 dimer, revealing the basis for the ability of unliganded C-Mad2, but not O-Mad2 or liganded C-Mad2, to form symmetric dimers. A Mad2 mutant that predominantly forms the C-C dimer is functional in vitro and in living cells. Finally, the Mad1-Mad2 core complex facilitates the conversion of O-Mad2 to C-Mad2 in vitro. Collectively, our results establish the existence of a symmetric Mad2 dimer and provide insights into Mad1-assisted conformational activation of Mad2 in the spindle checkpoint.

Show MeSH
Related in: MedlinePlus