The Tribbles family of pseudokinases recruits substrates to the ubiquitin ligase COP1 to facilitate ubiquitylation. CCAAT/enhancer-binding protein (C/EBP) family transcription factors are crucial Tribbles substrates in adipocyte and myeloid cell development. We found that the TRIB1 pseudokinase was able to recruit various C/EBP family members and that the binding of C/EBPβ was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBPα, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBPα in a pseudosubstrate-like manner. Crystallographic analysis and molecular dynamics and subsequent biochemical assays showed that C/EBP binding triggered allosteric changes that link substrate recruitment to COP1 binding. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation—by means of the activation loop and αC helix—and raise the possibility of small molecules targeting either the activation “loop-in” or “loop-out” conformations of Tribbles pseudokinases.

Tribbles假激酶家族募集底物至泛素连接酶COP1，以促进泛素化。CCAAT /增强子结合蛋白（C / EBP）家族转录因子是脂肪细胞和骨髓细胞发育中至关重要的Tribbles底物。我们发现TRIB1假激酶能够募集各种C / EBP家族成员，并且磷酸化减弱了C /EBPβ的结合。为了解释C / EBP募集的机制，我们解决了TRIB1与C /EBPα的复合晶体结构，这表明TRIB1相对于其无底物结构发生了显着的构象变化，并以伪底物状结合了C /EBPα。方式。晶体学分析，分子动力学和随后的生化分析表明，C / EBP结合触发了变构变化，将底物募集与COP1结合联系起来。这些发现为假激酶的调控提供了一个与真正的激酶调控显着相似的观点-通过激活环和αC螺旋-并提高了小分子靶向激活“环内”或“环外”构象的可能性。 Tribbles假激酶。