Substrate binding allosterically relieves autoinhibition of the pseudokinase TRIB1

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/EBP beta was attenuated by phosphorylation. To explain the mechanism of C/EBP recruitment, we solved the crystal structure of TRIB1 in complex with C/EBP alpha, which revealed that TRIB1 underwent a substantial conformational change relative to its substrate-free structure and bound C/EBP alpha 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 alpha C helix-and raise the possibility of small molecules targeting either the activation loop-in or loop-out conformations of Tribbles pseudokinases.