NMR-directed design of pre-TCRβ and pMHC molecules implies a distinct geometry for pre-TCR relative to αβTCR recognition of pMHC

The pre-T cell receptor (pre-TCR) guides early thymocytes through maturation processes within the thymus via interaction with self-ligands displayed on thymic epithelial cells. The pre-TCR is a disulfide-linked heterodimer composed of an invariant pre-TCR alpha (pT alpha) subunit and a variable beta subunit, the latter of which is incorporated into the mature TCR in subsequent developmental progression. This interaction of pre-TCR with peptide- major histocompatibility complex (pMHC) molecules has recently been shown to drive robust pre-TCR signaling and thymocyte maturation. Although the native sequences of beta are properly folded and suitable for NMR studies in isolation, a tendency to self-associate rendered binding studies with physiological ligands difficult to interpret. Consequently, to structurally define this critical interaction, we have re-engineered the extracellular regions of beta, designated as beta-c1, for prokaryotic production to be used in NMR spectroscopy. Given the large size of the full extracellular domain of class I MHC molecules such as H-K-b, we produced a truncated form termed K-b-t harboring properties favorable for NMR measurements. This system has enabled robust measurement of a pre-TCR-pMHC interaction directly analogous to that of TCR alpha beta-pMHC. Binding surface analysis identified a contact surface comparable in size to that of the TCR alpha beta-pMHC but potentially with a rather distinct binding orientation. A tilting of the pre-TCR beta when bound to the pMHC ligand recognition surface versus the upright orientation of TCR alpha beta would alter the direction of force application between pre-TCR and TCR mechanosensors, impacting signal initiation.