Abstract
T lymphocytes discriminate between healthy and infected or cancerous cells via T-cell receptor-mediated recognition of peptides bound and presented by cell-surface-expressed major histocompatibility complex molecules (MHCs). Pre-T-cell receptors (preTCRs) on thymocytes foster development of αβT lymphocytes through their β chain interaction with MHC displaying self-peptides on thymic epithelia. The specific binding of a preTCR with a peptide–MHC complex (pMHC) has been identified previously as forming a weak affinity complex with a distinct interface from that of mature αβTCR. However, a lack of appropriate tools has limited prior efforts to investigate this unique interface. Here we designed a small-scale linkage screening protocol using bismaleimide linkers for determining residue-specific distance constraints between transiently interacting protein pairs in solution. Employing linkage distance restraint-guided molecular modeling, we report the oriented solution docking geometry of a preTCRβ–pMHC interaction. The linkage model of preTCRβ–pMHC complex was independently verified with paramagnetic pseudocontact chemical shift (PCS) NMR of the unlinked protein mixtures. Using linkage screens, we show that the preTCR binds with differing affinities to peptides presented by MHC in solution. Moreover, the C-terminal peptide segment is a key determinant in preTCR–pMHC recognition. We also describe the process for future large-scale production and purification of the linked constructs for NMR, X-ray crystallography, and single-molecule electron microscopy studies.
Highlights
Adaptive T-cell-mediated immunity is driven by activation of T cells via their surface T-cell receptors (TCRs) [1,2,3,4]. αβT cells arise from T-lineage progenitors in the thymus that have been subjected to a series of selection events at discrete stages of intrathymic development, preserving useful specificities
Kb-t2 is a truncated version of Kb (derived from Kb-t in Ref [20]), which consists of the antigen presenting α1 and α2 domains of the presented by major histocompatibility complex molecules (pMHC) but lacks the α3 domain and the invariant β2 microglobulin subunit (β2M)
We suggest that the Pre-T-cell receptors (preTCRs)–pMHC interaction observed by NMR in case of the nonspecifically linked heterodimers is due to a supramolecular heterodimer–heterodimer interaction
Summary
Adaptive T-cell-mediated immunity is driven by activation of T cells via their surface T-cell receptors (TCRs) [1,2,3,4]. αβT cells arise from T-lineage progenitors in the thymus that have been subjected to a series of selection events at discrete stages of intrathymic development, preserving useful specificities while eliminating harmful ones [5,6,7,8]. Residues participating in the interaction between N15β and VSV8/Kb were identified by NMR cross-saturation or chemical shift perturbation (Fig. 1A), the docking orientation of the complex could not be unambiguously resolved from the experimental data.
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