Mechanistic insights into phosphorylation-driven activation and therapeutic inhibition of human ATR-ATRIP.

The ATR-ATRIP complex regulates the DNA replication stress and is targeted in cancer therapy, but the molecular mechanisms of its activation and inhibition have remained unclear. Here, we present high-resolution cryo-electron microscopy structures of the human ATR-ATRIP complex in distinct functional states, including two ATPγS-bound precatalytic states, a Chk1-bound catalytic state, a TopBP1-bound activated catalytic state and four inhibitor-bound states with gartisertib, berzosertib, ceralasertib and elimusertib. These structures show that ATPγS binding alone can prime the ATR kinase catalytic center for catalysis and position Chk1 without major conformational rearrangements. TopBP1 engages the ATR-ATRIP HEAT-repeat scaffold to reorganize the dimer into a near-symmetric conformation that promotes ATR-ATRIP autophosphorylation and efficient Chk1 phosphorylation. Notably, the phosphorylated ATR-ATRIP complex remains active toward Chk1 after TopBP1 dissociation while adopting an asymmetric conformation, indicating that catalytic activity is compatible with both near-symmetric and asymmetric conformations. In addition, inhibitor-bound structures reveal how potent and selective ATR inhibition is achieved through multipocket engagement and exploitation of active-site conformational plasticity. Together, our findings provide a structural framework for understanding multiple routes of ATR activation and inhibition, offering insights for developing next-generation ATR-targeted cancer therapies.