Disruption of R2TP-mediated PIKK maturation with de novo designed binders
- 1Spanish National Cancer Research Centre (CNIO), Structural Biology Programme, Melchor Fernandez Almagro 3, 28029 Madrid, Spain
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.
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BibTeX
@misc{gonzalezrodriguez2026disruption,
title = {{Disruption of R2TP-mediated PIKK maturation with de novo designed binders}},
author = {N. Gonzalez-Rodriguez and A. Lopez-Perrote and C. Reglero and J. Alegrio-Louro and M. Failde-Soler and C. Chacon-Sanchez and E. Blanco-Romero and E. Casajus-Pelegay and G. Lopez-Pernas and I. Hernandez and M. Murga and O. Fernandez-Capetillo and O. Llorca and R. Fernandez-Leiro},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.26.761954},
url = {https://www.biorxiv.org/content/10.64898/2026.10.26.761954v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - Disruption of R2TP-mediated PIKK maturation with de novo designed binders
AU - N. Gonzalez-Rodriguez
AU - A. Lopez-Perrote
AU - C. Reglero
AU - J. Alegrio-Louro
AU - M. Failde-Soler
AU - C. Chacon-Sanchez
AU - E. Blanco-Romero
AU - E. Casajus-Pelegay
AU - G. Lopez-Pernas
AU - I. Hernandez
AU - M. Murga
AU - O. Fernandez-Capetillo
AU - O. Llorca
AU - R. Fernandez-Leiro
PY - 2026
DA - 2026/10/26
PB - bioRxiv
DO - 10.64898/2026.10.26.761954
UR - https://www.biorxiv.org/content/10.64898/2026.10.26.761954v1
AB - 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.
ER -