Structural and mechanistic insights into the constitutive Themis–Grb2 complex in T cell signalling

Thymocyte selection is essential for establishing the T cell repertoire, maintaining self-tolerance and preventing autoimmunity. Themis, the archetypal member of a metazoan protein family defined by CABIT domains, centrally regulates this process by integrating T cell receptor (TCR) signalling. Themis has been proposed to constitutively partner with the multifunctional adaptor Grb2, yet the structural and mechanistic basis of this assembly has remained enigmatic. Here, we use Cryo-EM to reveal how the tandem CABIT domains and proline-rich sequence of Themis cooperatively engulf the C-terminal SH3 domain of Grb2, while the unbound domains of Grb2 remain poised to recruit additional binding partners. Furthermore, we uncover inherent interdomain flexibility in unbound Themis that resolves upon Grb2 binding. Structure-guided mutations abrogate the Themis–Grb2 interaction and fail to regulate the tyrosine phosphatase SHP-1 after TCR stimulation, recapitulating the phenotype of Themis-deficient cells. Our findings define the Themis–Grb2 complex as a dynamic structural hub in T cell signalling. Themis is a CABIT-domain containing protein that, in a constitutive complex with Grb2, regulates thymocyte selection downstream of TCR signalling. However, the structural basis of the formation of Themis/Grb2 complex has remained elusive. Here, the authors report Cryo-EM analysis of Themis, both unbound and in complex with Grb2, and functionally test the relevance of the identified putative interaction interfaces by mutagenesis, providing a structural blueprint of the molecular mechanisms underlying T cell signalling.