Structural basis for RAD18 regulation by MAGEA4 and its implications for RING ubiquitin ligase binding by MAGE family proteins

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Griffith-Jones, Simonne ; Álvarez, Lucía ; Mukhopadhyay, Urbi ; Gharbi, Sarah ; Rettel, Mandy ; Adams, Michael ; Hennig, Janosch ; Bhogaraju, Sagar:
Structural basis for RAD18 regulation by MAGEA4 and its implications for RING ubiquitin ligase binding by MAGE family proteins.
In: The EMBO Journal. Bd. 43 (2024) Heft 7 . - S. 1273-1300.
ISSN 1460-2075
DOI: https://doi.org/10.1038/s44318-024-00058-9

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Abstract

MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.