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.