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

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. MAGE proteins bind RING ubiquitin ligases and often promote pro-oncogenic pathways. Through structural and biochemical approaches, this study gives insight into how MAGEA4 binds and regulates the RING E3 RAD18, which is crucial for DNA repair.MAGEA4 utilizes a C-terminal peptide binding groove (PBG) to bind and regulate RAD18. The PBG is important for RING ligase binding in other MAGE proteins. MAGEA4 protects RAD18 from degradative autoubiquitination. The RAD18 RING and SAP domains interact together to mediate monoubiquitination of the substrate PCNA. The interaction mode is conserved in another MAGE/RING pair, MAGE-C2 and TRIM28. MAGEA4 partly displaces RAD6 from the key DNA repair E3 RAD18, thereby protecting it from degradative autoubiquitination.