Molecular mechanism of RUVBL1/2-TTT complex assembly by GNB1L
Abstract
Acting as dedicated co-chaperones, the AAA+ ATPases RUVBL1/2 and the TTT (TTI1-TTI2-TELO2) complex facilitate the maturation and assembly of phosphatidylinositol 3-kinase-related kinases (PIKKs), 270-470 kDa kinases critical for cellular homeostasis. However, the mechanisms governing RUVBL1/2-TTT assembly, the role of ATP hydrolysis, and the function of regulators like GNB1L remain unclear. Here we determined the cryo-EM structures of the full-length human TTT-RUVBL1/2-GNB1L complex in multiple functional states. The architecture reveals a 16-subunit assembly with three head-to-tail-linked TTT copies and a RUVBL1/2 hexamer in an asymmetric, mixed-nucleotide state. Unexpectedly, GNB1L adopts a tilted orientation to directly engage three Switch Loops of RUVBL1, tethering it to two ordered TELO2 protomers. We demonstrate that GNB1L, together with TTT, acts as a conformational hub that remodels the RUVBL1/2 ring to stimulate activity. Our findings establish that GNB1L physically integrates co-chaperone assembly while allosterically regulating its ATPase-driven chaperone function.
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