The Influence of Catalysis on Mad2 Activation Dynamics

Abstract

Mad2 is a key component of the spindle assembly checkpoint, a safety device ensuring faithful sister chromatid separation in mitosis. The target of Mad2 is Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mad2 binding to Cdc20 is a complex reaction that entails the conformational conversion of Mad2 from an open (O-Mad2) to a closed (C-Mad2) conformer. Previously, it has been hypothesized that the conversion of O-Mad2 is accelerated by its conformational dimerization with C-Mad2. This hypothesis, known as the Mad2-template hypothesis, is based on the unproven assumption that the natural conversion of O-Mad2 required to bind Cdc20 is slow. Here, we provide evidence for this fundamental assumption and demonstrate that conformational dimerization of Mad2 accelerates the rate of Mad2 binding to Cdc20. On the basis of our measurements, we developed a set of rate equations that deliver excellent predictions of experimental binding curves under a variety of different conditions. Our results strongly suggest that the interaction of Mad2 with Cdc20 is rate limiting for activation of the spindle checkpoint. Conformational dimerization of Mad2 is essential to accelerate Cdc20 binding, but it does not modify the equilibrium of the Mad2:Cdc20 interaction, i.e., it is purely catalytic. These results surpass previously formulated objections to the Mad2-template model and predict that the release of Mad2 from Cdc20 is an energy-driven process.

BUBR1 and Closed MAD2 (C-MAD2) Interact Directly to Assemble a Functional Mitotic Checkpoint Complex

“…For example, we realize that binding of either BUBR1 or C-MAD2 to the ␣C helix of C-MAD2 will block access of O-MAD2, thus formation of the MCC will naturally prevent "chronic C-MAD2 amplification," a question raised for the model suggesting that "liganded C-MAD2" catalyzes O3 C-MAD2 conversion (6,32). Moreover, as p31 comet , the negative regulator of the checkpoint, also binds to the ␣C helix of C-MAD2 (9, 10), switching binding partners at this region of C-MAD2 seems to hold significant importance in exerting control over both activation and silencing of the mitotic checkpoint.…”

“…In Vitro O3 C-MAD2 Conversion-This was carried out based on Simonetta et al (6) and illustrated in supplemental Fig. S3.…”

“…Although the predominant conformer in cells is O-MAD2, during mitosis the MAD1⅐C-MAD2 complex localized at unattached kinetochores catalytically converts the cytosolic pool of O-MAD2 into C-MAD2 (4,5). C-MAD2 released from kinetochores can form CDC20⅐C-MAD2 complexes that may further amplify the production of C-MAD2 by converting additional molecules of O-MAD2 (6).…”

Bimodal activation of BubR1 by Bub3 sustains mitotic checkpoint signaling

“…Unattached kinetochores stably bind a complex of Mad1 and Mad2 (5), which catalytically acts (8,10) to convert additional Mad2 molecules to an active form that binds Cdc20, thereby initiating mitotic checkpoint signaling. The other components of the MCC-BubR1, Bub3 (38), and Cdc20 (43)-are also recruited to unattached kinetochores (reviewed in ref.…”

“…This signaling pathway is initiated by a complex of Mad1 (mitotic arrest deficient 1) and Mad2 (mitotic arrest deficient 2) immobilized at unattached kinetochores (5). This complex then recruits a second Mad2 molecule (5-7) and catalyzes (8)(9)(10) its conformational change from open or N1 (inactive) to closed or N2 (active) (11,12) state. Closed Mad2 can bind Cdc20 (cell division cycle 20), the mitotic activator of the E3 ubiquitin ligase APC/C (anaphase promoting complex or cyclosome) that is responsible for advance to anaphase by its ubiquitination and subsequent proteasome-dependent degradation of cyclin B and securin.…”