A structurally altered D,L-amino acid TCRα transmembrane peptide interacts with the TCRα and inhibits T-cell activation in vitro and in an animal model

Protein-protein interactions in the membrane are pivotal for the cellular response to receptor-sensed stimuli. Recently, it has been demonstrated that an all-D-amino acids analogue of the TCRα transmembrane peptide (CP) is recruited to the TCR complex and inhibits T-cell activation in vitro and in vivo, similarly to the wild-type CP peptide. Here we investigated the relative contributions of the secondary structure of CP compared to its side chains in the association of CP with the TCR. We disrupted the secondary structure of CP by replacing two positive residues, needed for the interaction of CP with the TCR complex, by their D-enantiomers (2D-CP). Structure disruption was demonstrated by CD and FTIR spectroscopy, and molecular dynamics simulation in a bilayer environment. In vitro, 2D-CP colocalized with the TCR (visualized with confocal microscopy), immunoprecipitated with TCR but not MHC I, and inhibited T-cell activation. The peptide was effective also in vivo: it inhibited adjuvant arthritis in rats and delayed type hypersensitivity in BALB/c mice. Moreover, 2D-CP manifested greater immunosuppressive activity than wild-type CP, both in vivo and in vitro, which can be attributed to the greater solubility and resistance to degradation of 2D-CP. In molecular terms, these findings suggest that, under certain conditions, protein-protein interactions within the membrane might be more dependent on side chain interactions than on a specific secondary structure. The new altered secondary structure probably determines how the Lys and the Arg are positioned with respect to each other, so they can interact with the TM domain of the receptor. In clinical terms, the increased solubility and resistance to degradation of D-stereoisomers might be exploited in the targeted inactivation of pathogenic signaling pathways such as those arising from TCR-triggered activation of T-cells in immune-mediated disorders.