title: Structure of an intermediate conformer of the spindle checkpoint protein Mad2.
abstract: The spindle checkpoint senses unattached kinetochores during prometaphase and inhibits the anaphase-promoting complex or cyclosome (APC/C), thus ensuring accurate chromosome segregation. The checkpoint protein mitotic arrest deficient 2 (Mad2) is an unusual protein with multiple folded states. Mad2 adopts the closed conformation (C-Mad2) in a Mad1-Mad2 core complex. In mitosis, kinetochore-bound Mad1-C-Mad2 recruits latent, open Mad2 (O-Mad2) from the cytosol and converts it to an intermediate conformer (I-Mad2), which can then bind and inhibit the APC/C activator cell division cycle 20 (Cdc20) as C-Mad2. Here, we report the crystal structure and NMR analysis of I-Mad2 bound to C-Mad2. Although I-Mad2 retains the O-Mad2 fold in crystal and in solution, its core structural elements undergo discernible rigid-body movements and more closely resemble C-Mad2. Residues exhibiting methyl chemical shift changes in I-Mad2 form a contiguous, interior network that connects its C-Mad2-binding site to the conformationally malleable C-terminal region. Mutations of residues at the I-Mad2-C-Mad2 interface hinder I-Mad2 formation and impede the structural transition of Mad2. Our study provides insight into the conformational activation of Mad2 and establishes the basis of allosteric communication between two distal sites in Mad2.
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors: Mayuko
authors: M
authors: Hara
authors: Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors: Engin
authors: E
authors: Özkan
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390;
authors: Hongbin
authors: H
authors: Sun
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390 xuelian.luo@utsouthwestern.edu hongtao.yu@utsouthwestern.edu.
authors: Hongtao
authors: H
authors: Yu
authors: Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, TX 75390; xuelian.luo@utsouthwestern.edu hongtao.yu@utsouthwestern.edu.
authors: Xuelian
authors: X
authors: Luo
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002151:Calorimetry; D064199:Cdc20 Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D064246:Mad2 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008938:Mitosis; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary
chemical_list: D064199:Cdc20 Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D064173:Anaphase-Promoting Complex-Cyclosome
keywords: NMR; X-ray crystallography; allostery; conformational change; mitosis