Kinetic evidence for a ligand-binding-induced conformational transition in the T cell receptor

Abstract

Thermodynamics and kinetics of the interaction between T cell receptor specific for cytomegalovirus peptide (TCRCMV) and its specific ligand, pp65-HLA-A*0201 complex, were studied by surface plasmon resonance and stopped-flow methods. In the latter measurements, fluorescence resonance energy transfer (FRET) between fluorescently labeled reactants was used. Thermodynamic data derived from surface plasmon resonance measurements suggest that the complex formation is driven by both favorable enthalpy and entropy. Two reaction phases were resolved by the stopped-flow measurements. The rate constant of the first step was calculated to be close to the diffusion-controlled limit rate (3⅐10 5 to 10 6 M ؊1 ⅐s ؊1 ), whereas the second step's reaction rate was found to be concentration independent and relatively slow (2-4 s ؊1 at 25°C). These findings strongly suggest that the interactions between the TCR and its ligand, the peptide-MHC complex, proceed by a two-step mechanism, in which the second step is an induced-fit process, rate determining for antigen recognition by TCR.conformational changes ͉ T cell receptor-ligand interactions ͉ FRET ͉ stopped flow ͉ induced fit T cell receptor (TCR) association with its ligand, a peptide bound to molecules encoded by class I or II of the major histocompatibility complexes (pMHC), is the initial process in T cell activation (1-3). This process can induce different types of immune responses depending on the nature of the pMHC and the involved T cell (4-6). It is well established that despite the prevalent micromolar affinity, this interaction is characterized by such an exquisite sensitivity that even just a few antigenic pMHC molecules can be recognized on an antigen-presenting cell (APC) (7). At the same time, a TCR was shown to cross-react with different antigenic pMHC ligands (8 -10). Threedimensional structures determined for more than two dozen different TCR-pMHC complexes revealed similar diagonal orientations of TCR molecules with respect to their pMHC ligands (11,12). It was also found that two of the complementaritydetermining regions (CDRs) of the TCR, CDR1 and CDR2, interact predominantly with the MHC molecules, whereas the CDR3 interacts with exposed peptide residues. Importantly, significant conformational changes in the TCR, mainly in its CDR3, have been observed in crystallographic studies (10,13,14). These structural studies provide a static picture and require time-resolved analysis of the process to deepen the understanding of the molecular mechanism of antigen recognition by the TCR.Kinetic studies carried out to date have used the surface plasmon resonance (SPR) method, which has several deficiencies. First and foremost, its time resolution is limited. Essentially, results of all such studies were interpreted as bimolecular processes with a relatively slow rate constant of complex formation and a fast rate of its dissociation (3). This slow association rate was assigned to a conformational transition in the TCR binding site (15). This notion provided the basis f...

Low-affinity peptides and T-cell selection

“…A low affinity contact is considered more likely because, similar to peptide-devoid molecules, the flexibility of the binding groove helices is considerably enhanced when a peptide is only loosely bound [34]. Some resemblance to the first stage of the 'two-step' model of TCR binding to a pMHC that has been proposed previously [47,48] is obvious for the envisaged recognition, but remains a matter of speculation, and it is equally uncertain whether pMHC complexes interact as homo-or heterodimers with TCR in thymic selection processes [49]. In line with commonly held ideas, negative selection within the medulla would finally lead to deletion of all those T cells that are self-MHC-restricted, but still exhibit high affinity towards MHC molecules presenting any of the large number of self-peptides that can be displayed on mTEC [6].…”

Disparate Degrees of Hypervariable Loop Flexibility Control T-Cell Receptor Cross-Reactivity, Specificity, and Binding Mechanism

“…An NMR study of the D10 TCR reported greater flexibility of the CDR3α and CDR3β loops in the picosecond time scale 12 . Stopped-flow kinetic measurements have shown that the interaction of a cytomegalovirus peptide-specific TCR with its ligand is rate-limited by an induced-fit mechanism, but the location and magnitude of the associated structural changes that occur during binding is unknown 13 . Thermodynamic studies have suggested that a number of TCRs must undergo conformational changes during binding (reviewed in ref.…”

The TCR binding site does move

“…Even more intriguing and important, the study by Gakamsky et al (5) could provide a quantitative understanding of the difference between agonistic and antagonistic peptides presented by the same MHC. Another challenge for the future would be to measure the TCRpMHC interactions on the surface of living cells because that is where they are expressed and act in physiological situations.…”

“…This landmark study by Gakamsky et al (5) should be a basis for future work to unravel in further detail the interactions between TCRs and their ligands. TCRs always bind pMHC in a similar, relative diagonal orientation (2).…”