When the first structures of αβTCRs bound to MHC ligands appeared, the hope was that they would answer the question of why TCRs are confined to MHC ligands. We expected to see easily identifiable conserved interactions of the TCR CDR1 and CDR2 loops with the MHC helices, explaining the evolutionary connection between the TCR and MHC. It has taken 15 years and dozens of TCR-MHC structures to understand that the “rules of engagement” between TCRs and MHC ligands are much more complicated than originally imagined. Their interactions are not conserved in the conventional structural sense, but rather allow for considerably more flexibility, within the confines of certain general rules.
One of these rules has been well-established. In all structures solved so far, the TCR has a diagonal orientation on the MHC (reviewed in (Rudolph et al., 2006
). The CDR loops of the TCR usually occupy similar sites in the different structures, with the CDR2 loops interacting particularly with the MHC alpha helices and the very variable CDR3 loops centered on the peptide, while the CDR1 loops often contact both peptide and MHC. This rule applies for both MHCI and MHCII ligands and can be seen in the structure of the YAe62 TCR bound to either IAb
-p3K or Kb
-pWM. However, within this rule there is considerable latitude in the angle of engagement when viewed from above, in the pitch and yaw when viewed from the side or down the axis of the peptide binding groove (Bjorkman, 1997
; Rudolph et al., 2006
; Sethi et al.) and, in few extreme cases, involving some autoimmune TCRs, in the position of the TCR along the length of the peptide (Hahn et al., 2005
; Li et al., 2005
). Accordingly, there are some differences in the angle and yaw of the YAe62 TCR when bound to Kb
-pWM compared to IAb
With the accumulation of more structures of bound versus free TCRs, individual TCRs bound to different ligands and different TCRs using the same V element, it has also become clear that the general orientation of the TCR on the MHC involves considerable flexibility in the TCR CDR loops, especially the CDR3 loops (Gagnon et al., 2006
; Reiser et al., 2003
). The loops adjust to different ligands by changing not only the rotamers of their amino acid side chains, but also the conformation of their main chain backbones. This point is well-illustrated in the structures presented here. There are numerous differences in the rotamers of particular CDR amino acids of the YAe62 TCR when engaging its MHCI vs. MHCII ligand. Changes in backbone conformation of the CDR3 loops are particularly extensive.
The semi-conserved orientation of the TCR on MHC has raised the question of whether there are specific germline encoded V region amino acids selected evolutionarily to be linchpin positions for MHC interaction, thus imposing this orientation. The answer to this question has been harder to tease out from the structural data, since it requires comparison of a substantial set of structures involving different MHC ligands and TCRs all of which have the same TCR Vα and/or Vβ element. This requirement has only been met for a handful of V elements, such as those related to mouse Vβ8 and Vα4 discussed here and present in the YAe62 TCR. These V elements contain four amino acids that repeatedly have been observed to interact with the MHCII helices at the same locations (Dai et al., 2008
; Feng et al., 2007
; Maynard et al., 2005
). Those interactions are particularly well-illustrated in the YAe62 complexes with IAb
-p3K. We find the same amino acids in similar positions on the Kb
-pWM ligand and their mutation dramatically reduced YAe62 binding to both ligands. It is noteworthy that two of these amino acids are tyrosines (αY29 and βY48), whose interaction with the MHC does not require the precise geometry seen in H-bonds, but is dependent rather on the ability of their aromatic ring to create a substantial area of Van der Waals interaction, thus contributing to flexibility in the details of the contact. In both cases the approach of these amino acids to the MHC helix is facilitated by adjacent MHC amino acids lacking an interfering side chain.
Whether the presence of conserved germline encoded MHC interactions will extend to most other V elements will depend on many more new TCR-MHC structures and probably take many years to work out. It could be argued that, given the variability of the TCR angle, pitch and yaw on MHC, these germline encoded interactions will prove to be rare. However, we have seen a very high frequency of highly MHC crossreactive T cells, such as YAe-62, developing in single peptide mice that have limited negative selection (Huseby et al., 2005
). We argue that the processes of positive and negative selection in the thymus of normal animals select against these T cells, whose TCRs may display a full set of germline encoded interactions and therefore be likely to be strongly self-reactive. Rather, these processes select for TCRs whose somatically generated CDR3 loops interfere with some, but not all, of the possible germline interactions, steering the TCR specificity toward the peptide while preserving the TCR orientation on MHC via a much lower MHC affinity.
The most unexpected finding in the present study was a substantial disruption of the conventional Jα connection to Vα in the YAe62 TCR when bound to Kb
-pWM as compared to IAb
-p3K. This type of Vα domain conformational change has not been previously discussed. Nevertheless, our reexamination of previously published TCR structures turned up several examples of a similar altered conformation, one in a Vα and one in a Vβ element (Housset et al., 1997
; Mazza et al., 2007
; Reiser et al., 2000
; Reiser et al., 2002
). In each case the J region β strand had separated from the rest of the V element, preserving the critical Vα to Vβ interactions mediated by the two J region β strands. Also in each case, the flex point for this separation is the second glycine in the FGXG motif of the J element, raising the possibility that this motif in TCR J elements may be conserved to function as a swivel point for adjusting the interaction of Vα and Vβ. The result of these conformational changes is to alter the positions of the CDR1 and CDR2 loops of Vα relative to those of Vβ, influencing how these loops approach the MHC helices. This type of adjustment may be used in fine tuning the alignment of the CDR1 and CDR2 loops with particular regions of the MHC helices and as well as in accommodating MHC molecules with different distances between the two helices. Since the β-strands of J and V involved support the CDR3 loop, their repositioning also plays a role in the flexibility of this CDR3.
In the examples so far, this type of conformational change has been seen only in TCRs that interact with MHCI ligands. This may be coincidental, since there are many more TCR-MHCI than TCR-MHCII solved structures. Alternatively, the observation that the positions of the interactions of the amino acids conserved for MHC interaction are more consistent on MHCII ligands than on MHCI ligands may indicate that this additional flexibility in the TCR is more important for proper placement of these amino acids on MHCI ligands. Thus far, all of the examples of this conformational change in Vα involve TCRs constructed with a human Cα. Whether this Cα influences the ability of the Vα to undergo this change cannot be determined from these structures. However, we can say that the human Cα does not automatically induce this change, since there are many TCR structures with human Cα with attached Vα’s in the closed conformation, including that of the YAe62 TCR bound to IAb-p3K.
Unlike B cells that use antigen selected somatic mutation of their rearranged immunoglobulin genes to vastly increase their repertoire and fine tune their specificity for antigen, the T cell repertoire is limited by the set of initial rearrangements of TCR genes. Given that this repertoire is further constrained by the requirement for MHC reactivity and the processes of thymic positive and negative selection, there is a problem how the limited numbers of TCRs in any given animal can cope with the very large number of MHC-peptide combinations with which they may be confronted. This problem appears to have been solved in part by the relatively low affinities needed for TCR interaction with MHC-peptide to trigger response in T cells. In addition, the great flexibility of the CDR regions of TCRs allows a single TCR to bind a number of ligands. Here we have uncovered another phenomenon that allows recognition of more than one ligand by a single TCR, the potential to switch among three alternate conformations by disrupting the J to V interaction of either the Vα or Vβ domain.