Mutating a conserved motif in the activation loop of MEC1 (ATR) revealed the activation mechanism.
Major Finding: Mutating a conserved motif in the activation loop of MEC1 (ATR) revealed the activation mechanism.
Approach: Cryo–electron microscopy, genetic studies, and biochemical analyses were combined in this study.
Impact: Understanding the activation mechanism of ATR may enable targeting of this checkpoint protein.
The protein kinase ATR, the Saccharomyces cerevisiae homolog of which is MEC1, is a checkpoint protein that causes cell-cycle arrest and promotes DNA repair upon detection of DNA damage or replication fork stalling, making ATR an attractive target in cancers deficient in DNA damage repair. Using cryo–electron microscopy along with genetic and biochemical studies, Tannous, Yates, and colleagues discovered mechanisms by which MEC1 can be autoinhibited or activated. Mutating a highly conserved phenylalanine residue (F2244) in the DFD motif of MEC1′s activation loop to an alanine residue or a leucine residue boosted MEC1′s basal activity by approximately tenfold and twentyfold, respectively. The activity of MEC1F2244L was minimally affected by known activators; combined with its high basal activity, MEC1F2244L was therefore considered constitutively active. This lack of dependence on activators for activity enabled MEC1F2244L to rescue cell proliferation and DNA repair otherwise observed in MEC1 activator–deficient cells. Cryo–electron microscopy analyses of wild-type or F2244L-mutant MEC1 bound to the MEC1 partner DDC2 (the human homolog of which is ATRIP) along with the nonhydrolyzable ATP analogue AMP-PNP and Mg2+—in each case forming a dimer of Mg2+– and AMP-PNP–bound MEC1–DDC2 heterodimers—enabled determination of the structures of these tetrameric complexes at 3.8-Å and 2.8-Å resolution, respectively. Close inspection of the near-complete atomic models generated using the cryo–electron microscopy results combined with further experiments revealed how the activating F2244L mutation or activator binding altered the conformation of the activation loop, freeing MEC1 from autoinhibition and enabling it to exert its kinase activity. In summary, this work uncovers critical structural insights that—given the conservation of the DFD motif between MEC1 and ATR—may be exploitable for developing ATR targeted therapies for cancers with DNA-repair defects.
Tannous EA, Yates LA, Zhang X, Burgers PM. Mechanism of auto-inhibition and activation of Mec1ATR checkpoint kinase. Nature 2020 Nov 9 [Epub ahead of print].
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