Mechanism of allosteric inhibition of RUVBL1-RUVBL2 ATPase by the small molecule CB-6644

The RuvB-like protein 1/2 complex (RUVBL1/2) is a hetero-hexameric ATPase essential in several cellular processes, including chromatin remodeling and the assembly of mechanistic target of rapamycin complex 1 (mTORC1) and mTORC2. The aminopyrazolone CB-6644, a small molecule that allosterically inhibits RUVBL1/2, displays antitumor activity in animal models. However, its binding site and mechanism of action are not yet understood. Here, we applied cryoelectron microscopy (cryo-EM) to determine the ∼2.4 Å resolution structure of the RUVBL1/2 complex bound to ATP and CB-6644, which, together with additional biochemical and cryo-EM experiments, reveals that ATP binding to RUVBL1/2 induces large conformational changes that CB-6644 recognizes by interacting at the interface between two subunits. CB-6644 traps this ATP-bound conformation and prevents ATP hydrolysis. Interestingly, our results suggest a mechanism that couples the nucleotide state, the conformation of RUVBL1/2 domain II (DII), and the interaction of RUVBL1/2 with other proteins. Our findings reveal how ATP regulates RUVBL1/2 and how this is affected by CB-6644 binding.