姓名：张瑞豪  
自 强 不 息 | 厚 德 载 物  
日期：2024.08.9  
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Author information  
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Background  
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Main work: The researchers report cryoEM structures of two distinct full-length α/β TCR-CD3 complexes bound to  
their pMHC ligand, the cancer-testis antigen HLA-A2/MAGEA4 (230–239). They also determined cryoEM structures of  
pMHCs containing MAGEA4 (230–239) peptide and the closely related MAGEA8 (232–241) peptide in the absence of  
TCR.  
Theory base: TCRs are expressed as heterodimers of α/β or γ/δ chains in complex with three CD3 dimers (CD3εδ,  
CD3εγ, CD3ζζ) that are responsible for initiating downstream signaling. Sequence diversity in the variable domains,  
generated by V/D/J recombination similar to immunoglobulins, allow TCRs to discriminate their cognate pMHC  
molecules from the rest of the MHC-displayed proteome.  
Challenges: A landmark cryoelectron microscopy (cryoEM) structure of a full-length TCR-CD3 complex was recently  
described, as well as the first structure of a full-length affinity-enhanced TCRCD3 bound to pMHC. However, the  
application of cryoEM towards TCR-pMHC complex structure determination remains in its infancy. Furthermore,  
whether the naturally low affinity of the TCR-pMHC interaction precludes resolution of cryoEM structure remains  
unknown.  
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Research Implications: Elucidating the structural basis of antigen specificity is of particular relevance for tcr with  
therapeutic potential, as off-target responses to peptides presented on healthy cells can have dangerous consequences.  
Structural information can help improve the safety and efficacy of tcr-based therapeutics by facilitating the prediction of  
off-target peptides and the enhancement of structure-guided TCR-pMHC interactions.  
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PN45545 TCR-CD3  
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TCRs use three complementarity-determining  
regions (CDRs) on each chain to make contacts  
with the pMHC molecule. Almost all TCR  
structures have shown a canonical docking mode  
in which the CDR1 and CDR2 loops interact  
primarily with the MHC molecule and CDR3  
loops contact the MHC-embedded peptide,  
governing antigen recognition.  
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the presence of a lipid density situated between  
the TM helices of TCRβ, CD3γ, and CD3ζ  
subunits that they tentatively assigned as  
cholesteryl hemisuccinate (CHS) due to its  
matching shape features and its presence in  
purification buffer.  
The structure and arrangement of the TCR  
constant regions and CD3 subunits is nearly  
identical to previously published TCR-CD3  
complex. However, the elbow angle between  
TCR variable and constant regions is slightly  
different, likely reflecting their distinct Vα/Vβ  
sequences.  
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PN45545 TCR-CD3 complex structure supports the notion that  
the overall structure and assembly of TCR-CD3 is  
unaffected by differences in TCR variable region sequence.  
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TCR-CD3 complexes with HLA-A2/MAGEA4  
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The only notable differences when comparing the unligated  
and ligated PN45545 TCR-CD3 structures were present at the  
CDRs. However, they suggest cautious interpretation of  
these structural differences because the CDRs are not well  
defined in the PN45545 TCR-CD3 map calculated in the  
absence of pMHC.  
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The overall TCR-pMHC binding orientations of PN45545 and  
PN45428 follow the canonical docking polarity important  
for productive coreceptor binding.  
Despite their distinct docking angles, both PN45545 and  
PN45428 bind MAGEA4 pMHC such that the CD8αβ Ig  
domain C-termini would be oriented toward the T  
cellmembrane, favoring a cis configuration of TCR/CD3/CD8  
and trans binding of pMHC/CD8 as proposed previously. This  
observation illustrates how the geometric constraints of  
coreceptor binding can accommodate a range of  
TCR/pMHC binding angles.  
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TCR-CD3 complexes with HLA-A2/MAGEA4  
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Although their docking angles and CDR sequences  
are distinct, both TCRs have a binding footprint  
that is shifted toward the N-terminus of the  
MAGEA4 peptide.  
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The C-terminal portion of the peptide (E7-V10) is  
not contacted, suggesting it does not play a direct  
role in TCR recognition.  
Peptide contacts for both PN45545 and PN45428  
are mediated mainly by CDR3β. The mobility of  
the R6 side chain, noted previously in a crystal  
structure of MAGEA4 pMHC without TCR40,  
therefore appears to be critical for recognition of this  
MAGEA4 peptide antigen by different TCRs.  
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4 Structural discrimination between MAGEA4 and MAGEA8 peptides  
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HLA-A2/MAGE-A4(230–239) reactive TCRs  
have previously shown crossreactivity to a  
similar HLA-A2-restricted peptide derived  
from MAGEA8 residues 232–241. To assess  
the MAGEA4/MAGEA8 crossreactivities  
for PN45545 and PN45428, the TCRs were  
first expressed in primary human T cells and  
analyzed by flow cytometry with pMHC  
tetramer reagents.  
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They observed that the V2L mutant showed  
negligible binding to PN45545 and reduced  
binding to PN45428 relative to wild type  
MAGEA4 peptide. The T9S mutant peptide  
showed binding to both PN45545 and  
PN45428 at levels similar to MAGEA4.  
Therefore, the reduced affinity of the TCRs  
towards HLA-A2/MAGEA8 can be  
attributed to the V2L anchor residue  
substitution.  
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4 Structural discrimination between MAGEA4 and MAGEA8 peptides  
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The structures of MAGEA4 and MAGEA8 peptides  
were highly similar. The most notable difference  
occurs at peptide residue D4.  
The distinct D4 conformations can be attributed to the  
V/L substitution at position 2; the bulkier leucine side  
chain in MAGEA8 results in a slight remodeling of the  
peptide backbone that in turn favors the formation of  
the peptide D4/HLA R65 salt bridge.  
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4 Structural discrimination between MAGEA4 and MAGEA8 peptides  
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The structures of MAGEA4 and MAGEA8 pMHCs  
suggest that different conformations of peptide  
residue D4 are key for discrimination. Indeed, both  
TCRs make multiple contacts with the peptide D4 side  
chain, which adopts the down conformation.  
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Both TCRs interact directly with HLA-A2 α1 helix  
residue R65 through residues in CDR2β. Notably, R65  
frequently participates in TCR interactions as part  
of an HLA ‘restriction triad’ .  
Preferential binding by these TCRs for MAGEA4 is  
likely due to interactions that require the peptide D4 to  
be in a down conformation where it does not interact  
with HLA residue 65. Adoption of a MAGEA4-like  
peptide conformation by MAGEA8 would require  
disruption of the peptide D4-R65 salt bridge, which  
may be energetically unfavorable.  
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Discussion  
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The substitution of leucine by a conserved valine at position 2 of the anchoring residue can have a  
metastatic effect on the conformation of solvent-exposed peptide residues contacted by the TCR, thereby  
affecting recognition. This finding has important implications for the development of cancer vaccines that  
utilize mutated anchoring residues (typically introducing leucine at position 2) to improve peptide  
stability in the HLA groove, and confirms previous studies showing that anchoring residue modifications  
can affect TCR recognition.  
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They show how TCR binds to pMHC in the context of a full-length signaling complex containing the  
CD3 subunit, allowing assessment of the relative positions of the antigen and the t-cell membrane, which  
is not possible when using soluble tcr for structural determination. The researchers did not find any  
significant structural changes in the constant structural domain of the TCR or in the CD3 subunit induced  
by antigen attachment.  
Multivalent engagement of dimeric or tetrameric pMHC with multiple TCR-CD3 complexes is required to  
detect conformational changes in CD3ε50. pMHC triggering of the TCR is also thought to require the  
application of external mechanical forces. Finally, pMHC binding may variably induce dynamic  
changes in TCR-CD3, an effect that is difficult to observe from static cryostructures. Thus, the  
structural mechanisms by which pMHC activates TCR-CD3 signaling require further investigation.  
自 强 不 息 | 厚 德 载 物