Abstract
Regulated conformational changes of proteins are critical for cellular signal transduction. The spindle checkpoint protein Mad2 is an unusual protein with two native folds: the latent open conformer (O-Mad2) and the activated closed conformer (C-Mad2). During mitosis, cytosolic O-Mad2 binds to the Mad1-Mad2 core complex at unattached kinetochores and undergoes conformational activation to become C-Mad2. C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Here, we show that the conformational transition of Mad2 is regulated by phos- phorylation of S195 in its C-terminal region. The phospho-mimicking Mad2S195D mutant and the phospho-S195 Mad2 protein obtained using intein-mediated semisynthesis do not form C-Mad2 on their own. Mad2S195D fails to bind to Cdc20, a low-affinity ligand, but still binds to high-affinity ligands, such as Mad1 and MBP1, forming ligand-bound C-Mad2. Overexpression of Mad2S195D in human cells causes checkpoint defects. Our results indicate that Mad2 phosphor- ylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20 and establish that the conformational change of Mad2 is regulated by posttranslational mechanisms.
Original language | English (US) |
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Pages (from-to) | 19772-19777 |
Number of pages | 6 |
Journal | Proceedings of the National Academy of Sciences of the United States of America |
Volume | 107 |
Issue number | 46 |
DOIs | |
State | Published - Nov 16 2010 |
Keywords
- Conformational change
- Mitosis
- NMR
- Protein structure
- Spindle checkpoint
ASJC Scopus subject areas
- General