Peptide–MHC (pMHC) binding to a human antiviral T cell receptor induces long-range allosteric communication between pMHC- and CD3-binding sites

Abstract

T cells generate adaptive immune responses mediated by the T cell receptor (TCR)-CD3 complex comprising an αβ TCR heterodimer noncovalently associated with three CD3 dimers. In early T cell activation, αβ TCR engagement by peptide-major histocompatibility complex (pMHC) is first communicated to the CD3 signaling apparatus of the TCR-CD3 complex, but the underlying mechanism is incompletely understood. It is possible that pMHC binding induces allosteric changes in TCR conformation or dynamics that are then relayed to CD3. Here, we carried out NMR analysis and molecular dynamics (MD) simulations of both the α and β chains of a human antiviral TCR (A6) that recognizes the Tax antigen from human T cell lymphotropic virus-1 bound to the MHC class I molecule HLA-A2. We observed pMHC-induced NMR signal perturbations in the TCR variable (V) domains that propagated to three distinct sites in the constant (C) domains: 1) the Cβ FG loop projecting from the Vβ/Cβ interface; 2) a cluster of Cβ residues near the Cβ αA helix, a region involved in interactions with CD3; and 3) the Cα AB loop at the membrane-proximal base of the TCR. A biological role for each of these allosteric sites is supported by previous mutational and functional studies of TCR signaling. Moreover, the pattern of long-range, ligand-induced changes in TCR A6 revealed by NMR was broadly similar to that predicted by the MD simulations. We propose that the unique structure of the TCR β chain enables allosteric communication between the TCR-binding sites for pMHC and CD3.

The TCR is an allosterically regulated macromolecular machinery changing its conformation while working

“…However, relevant changes that control a proteins activity might be in protein dynamics. Several studies, have suggested that ligand binding can alter protein flexibility at distant sites, even in the absence of crystallographically observed structural changes …”

“…Deuterium/hydrogen exchange and NMR data have shown that the TCRαβ V and C regions are flexible. Upon pMHC binding this flexibility is reduced, and regions implicated in communication with CD3 show large structural changes.…”

“…These are the Cα AB and the Cβ FG loops, residues near the Cβ αA helix and the Cβ H3 and H4 helices (Figure ). Indeed, a molecular pathway of how changes at V cause changes at C has been suggested using molecular dynamics simulations . Thus, TCRαβ is allosterically regulated; binding of pMHC at one site (V regions) causes structural alterations and dynamic changes at other sites, namely those in contact with the CD3 subunits.…”

“…Secondly, structural changes within a single subunit or domain; called “true” conformational changes exist, similar to a bouncy rubber ball that changes its structure when hitting the floor. The structural changes in the TCRαβ ectodomains binding to pMHC are of this latest kind . Thus, in the TCR the rigid regions might not change their structure, but their relative orientation towards other regions, while other parts, such as the ectodomains or tails, might change their individual structures.…”

Stabilization of soluble high‐affinity T‐cell receptor with de novo disulfide bonds

“…Importantly, the identified stabilization motif in the constant domains had no effect on the affinity to the cognate pMHC and the molecule retained the specificity of antigen recognition, as proven with an unrelated MHC‐bound peptide. It would, however, be very interesting to study the activity of the proposed stabilized TCRs when introduced into the context of the cell‐bound T‐cell signaling complex, as it has been reported that the conformation of Cβ can critically affect cell signaling via the interaction with the components of the CD3 complex . As the stabilization motif did not adversely impact the expression level, heterodimerization properties, or the cognate pMHC antigen binding, the novel scaffold is a promising tool for further derivatization of the soluble TCR format, for example, mutagenesis toward functionalization of TCR constant domains for recognition of another antigen, as achieved for immunoglobulin constant domains .…”