PI3K-related kinases (PIKKs) are a family of giant protein kinases, characterized by extended N-terminal alpha-helical repeat regions and a conserved C-terminal kinase domain. Most members are involved in DNA and RNA quality control, for example ATM (ataxia-telangiectasia mutated) mediating double-strand DNA break repair, while specifically mTOR (mammalian target of rapamycin) controls cell growth. Despite their conserved domain organization, PIKKs assemble into diverse functional complexes, with or without additional partner proteins, with different oligomeric modes and overall complex architecture. The assembly of functional PIKK complexes depends on various chaperone systems, including the TTT co-chaperone complex comprising three protein subunits. TTT mediates interactions of the various nascent PIKKs with additional chaperones including Hsp90, but the mechanism of how TTT recognizes PIKKs and guides their assembly remains enigmatic. As mediators of crucial cellular functions, several PIKKs have been identified as relevant anti-cancer targets, and better understanding their assembly could provide novel paths to pharmaceutically targeting the PIKK family.
To determine features that mediate PIKK-TTT interactions we have used a comprehensive approach in cells and in vitro. We have established a cellular Bioluminescence Resonance Energy Transfer (BRET) assay to infer regions of PIKKs relevant to recognition by TTT by their ability to mediate proximity between PIKKs and TTT. Analysis of two different PIKK systems indicates the presence of at least two separate recognition sites. Furthermore, combining cryoEM and biochemistry, we study the dynamic nature of the TTT co-chaperone, how it acts as a transient binding platform for PIKKs, and its integration in the entire PIKK assembly machinery. We hypothesize that the interaction of TTT with PIKKs is based on multiple transient interactions, potentially regulated by cellular context. We aim to further explore this hypothesis by cellular, biophysical and cryoEM analysis of trapped states of TTT and PIKK interaction.