Righetto Germanna Lima, Yin Yanting, Duda David M, Vu Victoria, Szewczyk Magdalena M, Zeng Hong, Li Yanjun, Loppnau Peter, Mei Tony, Li Yen-Yen, Seitova Alma, Patrick Aaron N, Brazeau Jean-Francois, Chaudhry Charu, Barsyte-Lovejoy Dalia, Santhakumar Vijayaratnam, Halabelian Levon
Structural Genomics Consortium, University of Toronto, Toronto, Ontario M5G 1L7, Canada.
Department of Pharmacology and Toxicology, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
PNAS Nexus. 2024 Apr 10;3(4):pgae153. doi: 10.1093/pnasnexus/pgae153. eCollection 2024 Apr.
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.
损伤DNA结合蛋白1（DDB1）和CUL4相关因子12（DCAF12）作为Cullin4-RING E3连接酶（CRL4）复合物中的底物识别成分，能够识别C端双谷氨酸降解结构域，通过泛素蛋白酶体系统促进特定底物的降解。黑色素瘤相关抗原3（MAGEA3）和T复合物蛋白1亚基ε（CCT5）已被确定为DCAF12的细胞靶点。为了进一步表征DCAF12与MAGEA3和CCT5之间的相互作用，我们开发了一套生物物理和基于邻近性的细胞纳米BRET分析方法，结果表明MAGEA3和CCT5的C端降解结构域肽段在体外和细胞内均与DCAF12形成纳摩尔亲和力的相互作用。此外，我们在此报告了DDB1-DCAF12-MAGEA3复合物的3.17 Å冷冻电镜结构，揭示了负责C端降解结构域识别和结合的关键DCAF12残基。我们的研究提供了新的见解和工具，有助于发现靶向DCAF12的WD40重复结构域的小分子，用于未来蛋白酶靶向嵌合体的设计和开发。
