Structural and Biochemical Analysis of the CABIT1 Domain of THEMIS

T cells are important components of the adaptive immune system and develop through a selection process regulated by signaling through the T-cell receptor (TCR). Thymocyte- Expressed Molecule Expressed in Selection (THEMIS) is a TCR-proximal protein that modulates the activity of Shp1 phosphatase to influence TCR signaling during development. THEMIS has been shown to both activate and inhibit Shp1, but the molecular mechanisms of these functions are poorly understood. THEMIS contains two rare Cysteine All-Beta In THEMIS (CABIT) domains, the N-terminal of which interacts with Shp1 and is likely responsible for modulation of its phosphatase activity. Herein, we report the first crystal structure of the THEMIS CABIT1 domain. While a portion of the CABIT1 domain is poorly resolved, it appears to share the same overall fold observed in our recent CABIT2 crystal structure and AlphaFold predictions. We show that phosphorylation of the CABIT1 domain by LCK is required for association with SHP1 and that phosphorylated CABIT1 can protect the phosphatase domain of Shp1 from oxidation and inhibition by reactive oxygen species (ROS), which may serve as a mechanism by which THEMIS enhances Shp1 activity.