Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Phosphatidylinositol 3-kinase-related kinases (PIKKs), including ATM, ATR, DNA-PKcs, SMG1, TRRAP and mTOR, are regulators of gene expression, DNA damage response and metabolism. Their maturation depends on R2TP, a co-chaperone complex comprising the RUVBL1/2 AAA+ ATPase, RPAP3 and PIH1D1. While small-molecule RUVBL1/2 inhibitors have shown promise in cancer models, the presence of RUVBL1/2 in other complexes raises concerns about specificity. Here, we identified the RUVBL1/2-RPAP3 interface as a vulnerability, exploiting it using de novo designed protein binders that occupy the RPAP3-binding site. These subnanomolar-affinity binders dismantle R2TP, depleting levels of all PIKKs while sparing other RUVBL1/2-containing complexes, including chromatin remodelers INO80, SRCAP and TIP60. CryoEM, proteomic, biochemical and cellular analyses revealed that these binders outcompete RPAP3, trapping RUVBL1/2 in a catalytically inactive conformation. Our findings establish selective disruption of R2TP by artificial proteins as a strategy to control PIKK abundance.

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Publication Details

Journal
bioRxiv (Cold Spring Harbor Laboratory)
Published
2026-10-28
DOI
https://doi.org/10.64898/2026.10.26.761954
Primary Topic
Heat shock proteins research
Type
preprint

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preprint

Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Elena Blanco‐Romero, Óscar Fernández-Capetillo, Andrés López‐Perrote, Clara Reglero et al.
bioRxiv (Cold Spring Harbor Laboratory)
Heat shock proteins research
preprint

Disruption of R2TP-mediated PIKK maturation with de novo designed binders

Elena Blanco‐Romero, Óscar Fernández-Capetillo, Andrés López‐Perrote, Clara Reglero, Gema López-Pernas, Nayim González-Rodríguez, Óscar Llorca, Rafael Fernández-Leiro, Jaime Alegrio Louro, Ivó H. Hernández, Ester Casajús-Pelegay, Matilde Murga, Carlos Chacón-Sánchez, Marta Failde-Soler
preprint en

Abstract

Phosphatidylinositol 3-kinase-related kinases (PIKKs), including ATM, ATR, DNA-PKcs, SMG1, TRRAP and mTOR, are regulators of gene expression, DNA damage response and metabolism. Their maturation depends on R2TP, a co-chaperone complex comprising the RUVBL1/2 AAA+ ATPase, RPAP3 and PIH1D1. While small-molecule RUVBL1/2 inhibitors have shown promise in cancer models, the presence of RUVBL1/2 in other complexes raises concerns about specificity. Here, we identified the RUVBL1/2-RPAP3 interface as a vulnerability, exploiting it using de novo designed protein binders that occupy the RPAP3-binding site. These subnanomolar-affinity binders dismantle R2TP, depleting levels of all PIKKs while sparing other RUVBL1/2-containing complexes, including chromatin remodelers INO80, SRCAP and TIP60. CryoEM, proteomic, biochemical and cellular analyses revealed that these binders outcompete RPAP3, trapping RUVBL1/2 in a catalytically inactive conformation. Our findings establish selective disruption of R2TP by artificial proteins as a strategy to control PIKK abundance.

bioRxiv (Cold Spring Harbor Laboratory)
Spanish National Cancer Research Centre (ES)
European Regional Development Fund
Good health and well-being
Heat shock proteins research
DOIOpen Access PDFOpenAlex
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