Structural Features of the αβTCR Mechanotransduction Apparatus That Promote pMHC Discrimination

Abstract

The αβTCR was recently revealed to function as a mechanoreceptor. That is, it leverages mechanical energy generated during immune surveillance and at the immunological synapse to drive biochemical signaling following ligation by a specific foreign peptide-MHC complex (pMHC). Here, we review the structural features that optimize this transmembrane (TM) receptor for mechanotransduction. Specialized adaptations include (1) the CβFG loop region positioned between Vβ and Cβ domains that allosterically gates both dynamic T cell receptor (TCR)-pMHC bond formation and lifetime; (2) the rigid super β-sheet amalgams of heterodimeric CD3εγ and CD3εδ ectodomain components of the αβTCR complex; (3) the αβTCR subunit connecting peptides linking the extracellular and TM segments, particularly the oxidized CxxC motif in each CD3 heterodimeric subunit that facilitates force transfer through the TM segments and surrounding lipid, impacting cytoplasmic tail conformation; and (4) quaternary changes in the αβTCR complex that accompany pMHC ligation under load. How bioforces foster specific αβTCR-based pMHC discrimination and why dynamic bond formation is a primary basis for kinetic proofreading are discussed. We suggest that the details of the molecular rearrangements of individual αβTCR subunit components can be analyzed utilizing a combination of structural biology, single-molecule FRET, optical tweezers, and nanobiology, guided by insightful atomistic molecular dynamic studies. Finally, we review very recent data showing that the pre-TCR complex employs a similar mechanobiology to that of the αβTCR to interact with self-pMHC ligands, impacting early thymic repertoire selection prior to the CD4(+)CD8(+) double positive thymocyte stage of development.

Keywords: CD3; T cell receptor; antigen recognition; catch bond; kinetic proofreading; mechanosensor; pre-TCR; thymic development.

Figure 1

Models of the αβTCR and the pre-TCR complex. A side and top view of the assembly of the TCR ectodomain subunits on the surface of a cell membrane are illustrated. The αβTCR model was based on the following PDB codes: 1NFD, 1XMW, and 1JBJ; and the pre-TCR model was based on the PDB code 3OF6. The β chain is shown in blue for both the αβTCR and pre-TCR structures, the α chain is shown in red in the αβTCR and pTα in pink in the pre-TCR structure. For both the αβTCR and pre-TCR, the CD3ε chain is shown in cyan, the CD3γ chain in green, and the CD3δ chain in yellow. In the αβTCR, the CD3εγ heterodimer slots into the Cβ binding cleft in part created by the FG loop, and juxtaposed to the Cα domain. The white bracketed region in the side view illustrates the steric clash of the pTα domain with the CD3εγ heterodimer in this potential CD3 binding cleft. The structural crowding created due to the presence of the pre-TCR pTα chain is suggestive of an altered 3D configuration of the pre-TCR relative to the αβTCR. The white arrow highlights the presence of a significant gap generated between the pTα chain and the CD3εδ heterodimer as a consequence of the missing V domain and structural differences. Such a geometric alteration between the subunits may impose differing signaling requirements on the CD3εδ heterodimer in the pre-TCR vs. the αβTCR. The top view shows the absence of the Vα domain in the pre-TCR with the highly exposed pTα domain and CD3εδ subunits. Note that in the pre-TCR side view, the Vβ hydrophobic patch involves the exposed cell membrane-distal surface (upper left blue surface) buried by Vα in the TCR (34).

Figure 2

Rigidifying elements of the TCR. (A) The TCRαβ heterodimer (1OGA) compared with the Ig Fab (4JFX) fragments with equivalent domains represented in their respective CPK format. The dotted ellipse encloses the VβCβ interface. Note the more extensive interface in the TCR. A magnified view of the CβFG loop region is shown in yellow with the three conserved hydrophobic loop residues shown in blue and hydrogen bonds in red dashes. The 3₁₀ helix in the connector linking the Vβ and Cβ domains is also observed in this region and shown in brown. TCR–pMHC binding is thought to engage the FG loop, which would then mechanically push against this 3₁₀ helix and alter the overall TCR conformation. (B) Two views of CD3εγ are shown in which the linker region and several unstructured residues at the N termini of each domain have been omitted. CD3ε is depicted in blue and CD3γ in yellow. The structure below is rotated ~50° about the vertical axis relative to that on the top. The β strands are colored and labeled (blue for CD3ε and yellow for CD3γ). In the top structure, three pairs of main chain atoms involved in interdomain G strand hydrogen bonds are indicated with amide protons in gray and carbonyl oxygen atoms in red. In the bottom structure, the two pairs of disulfide-linked cysteine residues are shown as rods colored in magenta. Figure prepared using MOLMOL. (C) The heterodimeric CD3εγ subunit complex is illustrated, the CD3ε subunit is drawn in blue and the CD3γ subunit in yellow. Select interdomain hydrogen bonds are shown on the G strands as in (C). The cytoplasmic tails are illustrated vertically to depict receptor length, whereas physiologically, the tails may be associated with the inner leaflet of the plasma membrane. Each CD3 subunit contains an extracellular, transmembrane, and cytoplasmic domain. The CD3εγ extracellular domain structure was generated from the deposited PDB file 1XMW using PYMOL. The inset shows a representative model of the CD3εγ CxxC region containing an intramolecular disulfide bond at the N-terminus of the TM helices. Only the cysteine residues C82 and C85 of CD3γ are shown for clarity. (D) Comparison of the membrane-proximal stalk length found in cell surface molecules present on the surface of T cells. Both the TCRα and TCRβ chains have unusually long stalks relative to other cell surface receptors.

Figure 3

Evolutionary coupling between the TCR Cβ and CD3 gene products. In mammals, the TCR CβFG loop is significantly elongated and distinct CD3γ and CD3δ genes are present relative to the TCR genes found in birds, amphibians, reptiles, and bony fish. These mammalian adaptations during molecular speciation likely establish a more advanced immune system with greater sensitivity for pMHC recognition.

Figure 4

Force initiated TCR signaling. The force generated from the pMHC interaction with the TCR results in allosteric changes within the TCR subunits, most notably in the positioning of the TCR CβFG loop and rearrangement, and impacts the VαVβ module and quaternary αβTCR subunit changes. This restructuring through compression and tension forces (denoted by gray facing or opposing pairs of arrows) may alter the arrangement of the TCR TM domains and co-occur with changes in the membrane lipid composition. The ITAMs are thought to be released from the plasma membrane and become available to tyrosine kinase phosphorylation, thereby initiating T cell activation. Upon TCR–pMHC dissociation, the TCR returns to its initial disengaged state. In the illustration, the CD3ζ cytoplasmic tails are shown to be membrane associated, where the ITAM tyrosine residues are embedded into the membrane and consequently shielded from phosphorylation, as has been observed for the CD3ε cytoplasmic tail (–58). Other studies have demonstrated that the CD3ζ subunits exist in a constitutively phosphorylated state and therefore would not be associated with the lipid membrane as depicted (–61) (pMHC, orange; CβFG loop, magenta; TCR complex, other colors; lipid alteration, yellow circle).

Figure 5

Single-molecule TCRαβ–pMHC studies identify structural transitions. (A) The lifetime of αβTCR–pMHC bonds are analyzed by the single-molecule tether assay shown. “ΔX” represents the displacement of bead from the trap center. (B) Loading profile for measuring bond lifetime. Larger separation along the system path is shown through an increase in distance. A black dashed line represents an initial ramp phase, during this stage the tether is loaded to a fixed force and a green line illustrates the “Pre-transition dwell.” A signature structural transition is observed repeatedly, indicated by a green to blue line, and then followed by a “Post-transition dwell,” in blue. “Rupture,” shown by red line, is observed as an abrupt upward step. (C) Representative traces for VSV8/K^b at 10 pN for WT, ΔFG, and H57 Fab-bound TCRαβ. A commonly occurring transition and rupture is seen in the WT trace. An early transition is often detected for ΔFG, here occurring amid the initial ramp phase. H57 Fab traces do not show a transition nor a dwell for longer periods before rupture. The green baseline (see WT) illustrates observed low-amplitude motions.

Figure 6

N15 TCRαβ–pMHC force/lifetime curves, sensitivity indices and schematic diagram of low affinity and high affinity TCR binding mechanisms. (A) Force-bond lifetime plots for TCRβ WT, ΔFG, and H57 Fab-bound N15αβ. Catch bonds are observed for both VSV8 and L4 whereas slip bond character is seen for SEV9. Catch bonds peak at ~15 pN for WT and shift to lower force for ΔFG with significant reduction in bond lifetimes. Dramatic increases of catch bond lifetimes occur as a result of stabilization of the CβFG loop by the H57 Fab. (B) Sensitivity plots comparing TCRαβ–pMHC bond lifetime ratios of VSV8/SEV9 antigen for WT (solid, gray), H57 Fab (dashed, green), and ΔFG (dotted, purple). (C) Left, the TCR (multicolored) is in an unloaded, compact state with weak pMHC binding affinity depicted as loosely fitting pMHC (black). Subtle conformational motions may be occurring while gating potential interactions. Center, the TCR is in a loaded and more extended state, possessing high binding affinity resulting from significant structural rearrangements that create catch bonds. The pMHC is tightly bound to the TCR, resulting in the force generating engagement of the CβFG loop in a conformation binding the CD3εγ cleft for association (CD3ε, blue; CD3γ, yellow). Right, the TCR adopts a fully extended conformation where one or more conformational changes occur: CβFG loop disengagement, angular changes between the V to C domains and a weakened TCR–pMHC binding interface. For clarity, the CD3εδ heterodimer is not depicted.