Nanobodies identify an activated state of the TRIB2 pseudokinase.

Tribbles proteins (TRIB1-3) are pseudokinases that recruit substrates to the COP1 ubiquitin ligase. TRIB2 was the first Tribbles ortholog to be implicated as a myeloid leukemia oncogene, because it recruits the C/EBPα transcription factor for ubiquitination by COP1. Here we report identification of nanobodies that bind the TRIB2 pseudokinase domain with low nanomolar affinity. A crystal structure of the TRIB2-Nb4.103 complex identified the nanobody to bind the N-terminal lobe of TRIB2, enabling specific recognition of TRIB2 in an activated conformation that is similar to the C/EBPα-bound state of TRIB1. Characterization in solution revealed that Nb4.103 can stabilize a TRIB2 pseudokinase domain dimer in a face-to-face manner. Conversely, a distinct nanobody (Nb4.101) binds through a similar epitope but does not readily promote dimerization. In combination, this study identifies features of TRIB2 that could be exploited for the development of inhibitors and nanobody tools for future investigation of TRIB2 function.

Structural and biophysical characterization of Tribbles homolog 1.

Tribbles proteins are pervasive pseudokinases in cellular signaling. They play a major role in the differentiation of myeloid cells, hepatocytes and adipocytes, and more widely in immune function, metabolism and cancer. Like many other pseudokinases, an inherent lack of catalytic activity has meant that a specialized cadre of techniques has been required to investigate Tribbles function. A prerequisite to most in vitro biochemistry has been robust methods for purifying useful quantities of Tribbles protein, which can sometimes exhibit non-optimal behavior upon recombinant expression. For instance, structural studies of the Tribbles family have largely focused on TRIB1, in part because of more readily available protein. Here we describe methods we have developed to routinely produce milligram quantities of TRIB1, and specific considerations when employing TRIB1 protein for various downstream analyses. Namely, we describe preparation and crystallization of TRIB1 for structural studies, and using fluorescence polarization and isothermal titration calorimetry to analyze interactions with TRIB1. We hope that applying these considerations can facilitate further understanding of TRIB1 function, specifically, and can be selectively applied to improve studies of other Tribbles proteins and pseudokinases more generally.

Structure vs. Function of TRIB1-Myeloid Neoplasms and Beyond.

The Tribbles family of proteins-comprising TRIB1, TRIB2, TRIB3 and more distantly related STK40-play important, but distinct, roles in differentiation, development and oncogenesis. Of the four Tribbles proteins, TRIB1 has been most well characterised structurally and plays roles in diverse cancer types. The most well-understood role of TRIB1 is in acute myeloid leukaemia, where it can regulate C/EBP transcription factors and kinase pathways. Structure-function studies have uncovered conformational switching of TRIB1 from an inactive to an active state when it binds to C/EBPα. This conformational switching is centred on the active site of TRIB1, which appears to be accessible to small-molecule inhibitors in spite of its inability to bind ATP. Beyond myeloid neoplasms, TRIB1 plays diverse roles in signalling pathways with well-established roles in tumour progression. Thus, TRIB1 can affect both development and chemoresistance in leukaemia; glioma; and breast, lung and prostate cancers. The pervasive roles of TRIB1 and other Tribbles proteins across breast, prostate, lung and other cancer types, combined with small-molecule susceptibility shown by mechanistic studies, suggests an exciting potential for Tribbles as direct targets of small molecules or biomarkers to predict treatment response.