1. The Mad2 Spindle Checkpoint Protein Undergoes Similar Major Conformational Changes Upon Binding to Either Mad1 or Cdc20 
Xuelian Luo, Zhanyun Tang, Josep Rizo, Hongtao Yu 
Molecular Cell 
Volume 9, Issue 1, Pages (January 2002)
DOI: /S (01)00435-X

2. Figure 1 Mad1 Recruits Mad2 to Kinetochores in Prometaphase
(A) HeLa cells were transfected with the control (lanes 1 and 2) or the Mad1-targeting (lanes 3 and 4) siRNA duplexes for 36 hr, treated with thymidine (T) or nocodazole (N) for 18 hr to arrest cells at the G1/S boundary or mitosis, respectively, and blotted with antibodies against Mad1, Mad2, BubR1, and APC2.
(B) HeLa cells transfected with the control (left panel) or the Mad1-targeting (right panel) siRNA duplexes were fixed 48 hr after transfection and stained with α-Mad1.
(C) Mitotic HeLa cells transfected with the control (two left panels) or the Mad1-targeting (two right panels) siRNA duplexes were fixed and stained with α-Mad2 (in red) and CREST (in green).
(D) HeLa cells transfected with the Mad1-targeting siRNA duplex at various stages of mitosis were fixed and stained with CREST. The lagging kinetochores are indicated.
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)

3. Figure 2
Mad1 Is Required for Proper Signaling of the Mitotic Checkpoint
(A) HeLa cells were transfected with the control (left panels) or the Mad1-targeting (right panels) siRNA duplexes for 36 hr. The cells in log phase (top panels) or treated with nocodazole for 18 hr (bottom panels) were analyzed by FACS. The peaks corresponding to 2N and 4N DNA contents are labeled.
(B) The control or Mad1 RNAi cells were treated with nocodazole for 18 hr and stained with Hoeschst The cell (top panels) and nuclear (bottom panels) morphology of these live cells were directly visualized with an inverted fluorescence microscope. The multinucleated cells and cells with abnormal nuclear morphology are indicated by arrows.
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)

4. Figure 3 Identification of Mad2 Binding Peptides
(A) Schematic drawing of the structural motifs of Cdc20 and alignment of the Mad2 binding sequences of the Cdc20 proteins from various organisms (Hs, Homo sapiens; Dm, Drosophila melanogaster; Sc, Saccharomyces cerevisiae; and Sp, Schizosaccharomyces pombe). The key Mad2 binding residues are colored in red.
(B) Sequence alignment of the Mad2 binding sequences of the Mad1 proteins from various organisms. The key Mad2 binding residues are colored in red.
(C) Sequence alignment of ten Mad2 binding peptides (MBPs) identified using phage display and the Mad2 binding motifs of the human Mad1 and Cdc20 proteins. The consensus motif of MBPs is shown above. The critical binding elements are colored in red.
(D) Incubation of APC isolated from interphase Xenopus egg extracts (I-APC) with human Cdc20 greatly stimulates the activity of APC (compare lanes 1 and 2). The purified ΔN10-Mad2 protein inhibits the activity of APCCdc20 using cyclin B1 as a substrate (compare lanes 2 and 3). Addition of 100 μM MBP1 blocks the ability of Mad2 to inhibit APCCdc20 (lane 5) while a peptide containing the MBP1 sequence in reverse (MBP1-rev) has no effect (lane 4).
(E) The Mad1465–584 fragment also blocks the ability of Mad2 to inhibit APCCdc20 at high concentrations (10–20 μM) (compare lane 3 with lanes 4–9).
(F) FACS analysis of HeLa cells cotransfected for 36 hr with a plasmid encoding Mad2, together with plasmids encoding MBP1-rev-GFP (left panel), MBP1-GFP (central panel), or full-length Mad1 (right panel).
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)

5. Figure 4 Binding of Cdc20P1, Mad1P1, and MBP1 to Mad2 Monitored by NMR
(A) Overlay of the 1H-15N HSQC spectra of the free ΔN10-Mad2 (in black) and the ΔN10-Mad2-Cdc20P1 complex (in red).
(B) Overlay of the 1H-15N HSQC spectra of the free ΔN10-Mad2 (in black) and the ΔN10-Mad2-Mad1P1 complex (in cyan).
(C) Overlay of the 1H-15N HSQC spectra of the ΔN10-Mad2-Cdc20P1 complex (in red) and the ΔN10-Mad2-Mad1P1 complex (in cyan).
(D) Overlay of the 1H-15N HSQC spectra of the free ΔN10-Mad2 (in black) and the ΔN10-Mad2-MBP1 complex (in red).
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)

6. Figure 5
Solution Structure of Mad2-MBP1 and Comparison with that of the Free Mad2
(A) Stereo-view of the overlaid backbone traces of the 25 final NMR structures of human Mad2 in complex with MBP1. The β strands are shown in blue; α helices in green; and the loops in gray. MBP1 is colored in red. Generated with the program MOLMOL (Koradi et al., 1996).
(B) Ribbon drawing of the free (left) and ligand-bound (right) Mad2 structures. The β strands are shown in blue; α helices in green; and the loops in cyan. MBP1 is colored in red. The structural elements of Mad2 that undergo major changes upon peptide binding are colored yellow. The strands are numbered 1–8 while the helices are labeled A–C in the free Mad2 structure. MBP1 is labeled as β1′ in the Mad2-MBP1 complex. The rest of the secondary structure elements are labeled in a manner similar to the free Mad2 with the exception of β9, which is unstructured in free Mad2.
(C) Same as (B) but rotated 90° along the vertical (y) axis. Generated with the programs Molscript (Kraulis, 1991) and Raster3D.
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)

7. Figure 6 The Ligand Binding Site of Mad2
The color scheme of the ribbon drawings is the same as used in Figure 5. The Mad2 residues involved in the binding pockets are shown as gray ball-and-stick models and labeled. The MBP1 residues are also shown as ball-and-stick models and colored in yellow.
(A–E) The binding pockets for the residues at the P1–P5 positions of MBP1 and other Mad2 ligands.
(F) The putative binding pockets for the prolines of MBP1. Though no NOEs were identified between these prolines and the aromatic residues, the prolines are in close proximity to this site. Generated with the programs Molscript and Raster3D.
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)

8. Figure 7
Binding of Cdc20 or Mad1 Induces a Conformational Change of Mad2 Similar to that Caused by MBP1 Binding
(A) Ribbon drawing of Mad2-MBP1 with W100 and V197 shown as ball-and-stick models.
(B) Selected regions of a 3D 1H-15N NOESY-HSQC spectrum acquired on the 15N-Mad2-Cdc20P1 sample and a 3D 1H-13C NOESY-HSQC spectrum acquired on a Mad2-13C,15N-Cdc20P1 sample. Intermolecular NOEs from L133 of Cdc20 to W167 of Mad2 are labeled.
(C–E) Selected regions of 3D 1H-15N NOESY-HSQC spectra acquired on 15N-Mad2-Cdc20P1 (C), 15N-Mad2-Mad1P1 (D), or 15N-Mad2-MBP1 (E) complexes, showing NOEs from the methyl groups of V197 to the amide protons of R99, V197, and A198.
(F) A model for the formation of the Mad2-Cdc20-containing checkpoint complex(es). See Discussion for details.
Molecular Cell 2002 9, 59-71DOI: ( /S (01)00435-X)