Substrate binding allosterically relieves autoinhibition of the pseudokinase TRIB1

Abstract

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.

The pseudokinase TRIB 1 toggles an intramolecular switch to regulate COP 1 nuclear export

“…C Quantification of the average ratio of nuclear/cytoplasmic fluorescence of GFP-COP1 constructs in (A) and (B). Alternatively, a conformational change in the TRIB1 pseudokinase domain induced by C/EBPa binding (Jamieson et al, 2018) could alter the ability of TRIB1 to form interactions necessary for its localization to PML bodies. are shown for three independent experiments where 50 individual cells per experiment were analyzed.…”

“…Several studies have shown that mutations in the pseudokinase domain affect interactions between Tribbles and its binding partners. Recently, a crystal structure of TRIB1 in complex with a C/EBPa peptide revealed the binding site for C/EBPa within the C-lobe of the TRIB1 pseudokinase domain (Jamieson et al, 2018). A mutation within the HRD motif in the Drosophila Tribbles homolog ablates its interactions with Akt and the C/EBP homolog Slbo (Masoner et al, 2013;Das et al, 2014).…”

“…A mutation within the HRD motif in the Drosophila Tribbles homolog ablates its interactions with Akt and the C/EBP homolog Slbo (Masoner et al, 2013;Das et al, 2014). We also introduced two point mutations in TRIB1 that had been shown to ablate interaction with C/EBPa (H168D, F293E) seen in the crystal structure (Jamieson et al, 2018). Although TRIB1 is catalytically inactive (Murphy et al, 2015), these mutations might bias the pseudokinase domain to adopt different conformations and thereby affect the ability of TRIB1 to interact with its binding partners.…”

“…Although TRIB1 is catalytically inactive (Murphy et al, 2015), these mutations might bias the pseudokinase domain to adopt different conformations and thereby affect the ability of TRIB1 to interact with its binding partners. Recently, a crystal structure of TRIB1 in complex with a C/EBPa peptide revealed the binding site for C/EBPa within the C-lobe of the TRIB1 pseudokinase domain (Jamieson et al, 2018). We generated point mutations in the pseudoactive site of TRIB1 corresponding to those analyzed in Drosophila Tribbles (D205N) and in murine TRIB2 (K207R) ( Fig EV3A).…”

“…We generated point mutations in the pseudoactive site of TRIB1 corresponding to those analyzed in Drosophila Tribbles (D205N) and in murine TRIB2 (K207R) ( Fig EV3A). We also introduced two point mutations in TRIB1 that had been shown to ablate interaction with C/EBPa (H168D, F293E) seen in the crystal structure (Jamieson et al, 2018). None of these mutations, however, caused a significant change in the ability of TRIB1 to promote COP1 nuclear localization ( Fig EV3B-E).…”