Molecular dynamics simulations reveal canonical conformations in different pMHC/TCR interactions

The major defense system against microbial pathogens in vertebrates is the adaptive immune response and represents an effective mechanism in cancer surveillance. T cells represent an essential component of this complex system. They can recognize myriads of antigens as short peptides (p) originated from the intracellular degradation of foreign proteins presented by major histocompatibility complex (MHC) proteins. The clonotypic T-cell antigen receptor (TCR) is specialized in recognizing pMHC and triggering T cells immune response. It is still unclear how TCR engagement to pMHC is translated into the intracellular signal that initiates T-cell immune response. Some work has suggested the possibility that pMHC binding induces in the TCR conformational changes transmitted to its companion CD3 subunits that govern signaling. The conformational changes would promote phosphorylation of the CD3 complex ζ chain that initiates signal propagation intracellularly. Here, we used all-atom molecular dynamics simulations (MDs) of 500 ns to analyze the conformational behavior of three TCRs (1G4, ILA1 and ILA1α1β1) interacting with the same MHC class I (HLA-A*02:01) bound to different peptides, and modelled in the presence of a lipid bilayer. Our data suggest a correlation between the conformations explored by the β-chain constant regions and the T-cell response experimentally determined. In particular, independently by the TCR type involved in the interaction, the TCR activation seems to be linked to a specific zone of the conformational space explored by the β-chain constant region. Moreover, TCR ligation restricts the conformational space the MHC class I groove

Proximal changes in signal transduction that modify CD8+ T cell responsiveness in vivo

The antigen dose conditions the functional properties of CD8+ T cells generated after priming. At relatively low antigen doses, efficient memory T cells may be generated, while high antigen doses lead to tolerance. To determine the mechanisms leading to such different functional outcomes, we compared the proximal TCR signal transduction of naive cells, to that of memory or high-dose tolerant cells generated in vivo. In vivo activation led to the constitutive phosphorylation of CD3 4 , recruiting Zap70, in both memory and tolerant cells. In tolerant cells, these phenomena were much more marked, the CD3 4 and ´ chains no longer associated, and the Src kinases p56Lck and p59Fyn were inactive. Therefore, when the antigen load overcomes the capacities of immune control, a new mechanism intervenes to block signal transduction: the recruitment of Zap70 to CD3 4 becomes excessive, leading to TCR complex destabilization, Src kinase dysfunction, and signal arrest

Mutation of tyrosine 492/493 in the kinase domain of ZAP-70 affects multiple T-cell receptor signaling pathways.

The protein-tyrosine kinase ZAP-70 is implicated, together with the Src kinase p56lck, in controlling the early steps of the T-cell antigen receptor (TCR) signaling cascade. To help elucidate further the mechanism by which ZAP-70 regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited TCR-induced activation of nuclear factor of activated T cells by interfering with both intracellular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp36-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type ZAP-70 induced constitutive activation of nuclear factor of activated T cells. The ZAP-70 mutant studied here could be phosphorylated on tyrosine when associated to the TCR ζ chain and was able to bind p56lck. This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the Src homology domain 2-mediated association of p56lck with ZAP-70. Our data are most consistent with a model in which recruitment to the TCR allows ZAP-70 autophosphorylation and binding to p56lck, which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively control phosphorylation of its substrates and lead to gene activation

Molecular dynamics simulations reveal membrane lipid interactions of the full-length lymphocyte specific kinase Lck

The membrane-bound lymphocyte-specific protein-tyrosine kinase (Lck) triggers T cell antigen receptor signalling to initiate adaptive immune responses. Despite many structure–function studies, the mode of action of Lck and the potential role of plasma membrane lipids in regulating Lck’s activity remains elusive. Advances in molecular dynamics simulations of membrane proteins in complex lipid bilayers have opened a new perspective in gathering such information. Here, we have modelled the full-length Lck open and closed conformations using data available from different crystalographic studies and simulated its interaction with the inner leaflet of the T cell plasma membrane. In both conformations, we found that the unstructured unique domain and the structured domains including the kinase interacted with the membrane with a preference for PIP lipids. Interestingly, our simulations suggest that the Lck-SH2 domain interacts with lipids differently in the open and closed Lck conformations, demonstrating that lipid interaction can potentially regulate Lck’s conformation and in turn modulate T cell signalling. Additionally, the Lck-SH2 and kinase domain residues that significantly contacted PIP lipids are found to be conserved among the Src family of kinases, thereby potentially representing similar PIP interactions within the family