How Does the CRL3 Ubiquitin Ligase Dynamically Regulate Substrate Degradation?

Protein ubiquitination plays a central role in the life processes of eukaryotic cells. Cullin-RING E3 ubiquitin ligases (CRLs), as key components of the ubiquitin-proteasome system (UPS), represent the largest family of E3 enzymes in mammalian cells. They are responsible for the specific recognition, ubiquitination, and degradation of substrate proteins, thereby precisely regulating various physiological processes. As an important subtype, CRL3 specifically utilizes BTB domain-containing proteins as substrate adaptors. The human genome encodes over 180 BTB proteins, and CRL3 complexes are widely involved in critical biological events such as glucose and lipid metabolism, cancer development, cell cycle progression, and development. However, due to the structural complexity of CRL3, its molecular architecture and catalytic mechanisms remain largely unknown.

On February 8, 2024, a research team led by Professor Sun Lei and Professor Chen Zhenguo from the Institute of Biomedical Sciences at Fudan University, in collaboration with Nobel laureate Professor Bruce Beutler from the University of Texas Southwestern Medical Center, published a study titled “Dynamic molecular architecture and substrate recruitment of cullin3–RING E3 ligase CRL3 KBTBD2” in Nature Structural & Molecular Biology. Using the specific recruitment of p85α by CRL3_KBTBD2 as a model, this study systematically elucidates the dynamic structural changes and functional mechanisms of this E3 ligase during its catalytic cycle.

1. How Does CRL3 KBTBD2 Regulate the Insulin Signaling Pathway?

p85α is a regulatory subunit of the PI3Kα complex and, together with the catalytic subunit p110α, regulates insulin signal transduction. KBTBD2, a newly identified regulator of p85α, leads to p85α accumulation when deficient in mouse models, causing disruptions in insulin signaling. This results in phenotypes such as growth retardation, insulin resistance, type II diabetes, and even death. The study demonstrates that KBTBD2 assembles with CUL3 and RBX1 to form the CRL3_KBTBD2 complex, which catalyzes the ubiquitination and degradation of p85α, thereby playing a critical role in metabolic homeostasis.

2. What Dynamic Structural States Are Involved in the Catalytic Cycle of CRL3 KBTBD2?

This study successfully resolved high-resolution cryo-EM structures of CRL3 KBTBD2 in multiple functional states, including self-assembly, substrate recruitment (bound to p85α), activation (NEDD8 modification), deactivation (bound to CSN), and substrate receptor exchange (bound to CAND1), among seven distinct states. This constitutes the most complete model of the CRL3 catalytic cycle to date.

Notably, KBTBD2 itself possesses dimerization capability, enabling CRL3 KBTBD2 to form higher-order structures such as tetramers, hexamers, and even octamers. The enzyme’s active site remains shielded in the absence of substrate. Upon binding to factors such as p85α, NEDD8, or CSN, the complex gradually dissociates into an active dimeric form, exposing the substrate-binding and catalytic sites.

3. How Does CRL3 Specifically Recognize and Recruit Full-Length Substrate Proteins?

Elucidating the substrate recognition mechanism of the CRLs family has long been a major challenge. This study systematically reveals, for the first time, the interaction between CRL3_KBTBD2 and full-length p85α protein: KBTBD2 utilizes its BTB-BACK and Kelch domains to form multi-interface interactions with p85α, providing high affinity and specificity for substrate recognition. This work fills a gap in the structural mechanism of CRLs recruiting full-length substrates and offers new perspectives for understanding the substrate selectivity of E3 ligases.

4. What Are the Implications of This Study for Ubiquitination System Research?

This study proposes for the first time a dynamic conformational change model of CRL3 KBTBD2 during its catalytic cycle, systematically explaining its mechanism of substrate recruitment and ubiquitination through structural reorganization. This not only deepens the understanding of CRL3 function but also provides a structural basis for drug development targeting CRLs.

It is worth noting that the same issue of Nature Structural & Molecular Biology features a "Ubiquitination" special topic, publishing multiple related studies and commentaries, reflecting the continued activity and importance of this field in mechanistic exploration and clinical applications.

Conclusion

The ubiquitin-proteasome system plays a central role in maintaining protein homeostasis and cellular function, and its dysregulation is closely linked to various major diseases. Currently, drug development targeting this system has made substantial progress in cancer therapy, including proteasome inhibitors, deubiquitinating enzyme inhibitors, and ubiquitinating enzyme modulators, among other compounds. Future research will continue to deepen the understanding of the regulatory mechanisms of this system, promoting the development of more selective and efficient therapeutic strategies.

Product Information