Journal Article

Probing the CRL4 DCAF12 interactions with MAGEA3 and CCT5 di-Glu C-terminal degrons

Seitova, Alma, Brazeau, Jean-Francois, Loppnau, Peter, Barsyte-Lovejoy, Dalia, Santhakumar, Vijayaratnam, Patrick, Aaron N, Vu, Victoria, Li, Yanjun, Szewczyk, Magdalena M, Duda, David M, Mei, Tony, Li, Yen-Yen, Righetto, Germanna Lima, Zeng, Hong, Halabelian, Levon, Chaudhry, Charu, Yin, Yanting

2024

Overview

Damaged DNA-binding protein-1 (DDB1)- and CUL4-associated factor 12 (DCAF12) serves as the substrate recognition component within the Cullin4-RING E3 ligase (CRL4) complex, capable of identifying C-terminal double-glutamic acid degrons to promote the degradation of specific substrates through the ubiquitin proteasome system. Melanoma-associated antigen 3 (MAGEA3) and T-complex protein 1 subunit epsilon (CCT5) proteins have been identified as cellular targets of DCAF12. To further characterize the interactions between DCAF12 and both MAGEA3 and CCT5, we developed a suite of biophysical and proximity-based cellular NanoBRET assays showing that the C-terminal degron peptides of both MAGEA3 and CCT5 form nanomolar affinity interactions with DCAF12 in vitro and in cells. Furthermore, we report here the 3.17 Å cryo-EM structure of DDB1-DCAF12-MAGEA3 complex revealing the key DCAF12 residues responsible for C-terminal degron recognition and binding. Our study provides new insights and tools to enable the discovery of small molecule handles targeting the WD40-repeat domain of DCAF12 for future proteolysis targeting chimera design and development.